BACTERIA. BY DR. ANTOINE MAGNIN, \| LICENTIATE OF NATURAL SCIENCES; CHIEF OF THE PRACTICAL LABORS IN NATURAL HISTORY TO THE FACULTY OF MEDICINK OF LYONS; LAUREATE OP THE FACULTY OF MEDICINE OF PARIS (SILVER MEDAL, 1876); GENERAL SECRETARY OF THE BOTANICAL SOCIETY OF LYONS; MEMBER OF THJi BOTANICAL SOCIETY OF FRANCE; ETC. AND GEORGE M. STERNBERG, M.D., F.R.M.S., MAJOR AND SURGEON U. 8. ARMY; MEMBER OF THE BIOLOGICAL SOCIETY OF WASH- INGTON? LATE MEMBER OF THE HAVANA YELLOW FEVER COMMISSION OF TUB NATIONAL BOARD OF HEALTH; CORRESPONDING MEMBER OF THE EPIDEMIOLOGICAL SOCIETY OF LONDON} ETC. NEW YORK: WILLIAM WOOD AND COMPANY, 56 AND 58 LAFAYETTE PLACE. 1884. Copyright, 1880, BY GEORGK M. STERNBERG. Copyright, 1883, BY GEORGE M. STERNBERG. (ZTaminriiJge : PRINTED BY JOHN WILSON AND SON, UNIVERSITY 1'KESS. *M G PREFACE. THE work of Dr. Magnin, which was published in Paris in 1878 and translated by the writer in 1880, gave an admirable resume of our knowledge of the Bac- teria at the date of its publication. But very consid- erable progress has been made since, especially as regards methods of manipulation, the comparative value of various chemical reagents as "germicides" and anti- septics, and the role of the Bacteria^ in infectious.,. dis- eases. With a view to keeping the work fully up to the progress of science in this direction, the writer has added a chapter upon each of these subjects, and one upon " Bacteria in Surgical Lesions " (Parts Third, Fourth, Fifth, and Sixth). His name, therefore, appears upon the titlepage as one of the authors of the present volume. It has not been considered necessary, however, to rewrite the chapters on Morphology and Physiology (Parts First and Second). It is true that the classification of Cohn, which was very properly adopted by Professor Magnin, is only provisional, and that certain recently discovered pathogenic species are not included. But these will receive attention in Part Fifth of the present volume ; and it would be pre- mature to attempt a natural and permanent classifica- tion of these minute plants, which are now engaging the attention of numerous investigators in all parts of iv PREFACE. the civilized world. For the present we probably can- not do better than to adhere to the artificial classifica- tion, based upon morphological characters alone, which Cohn has given us. It must be remembered, however, that SPHERO- BACTERIA — micrococci — are not always round; that there is no well-defined line of demarcation between the MICRO-BACTERIA and the DESMOBACTERIA, between the genus BACTERIUM and the genus BACILLUS, or be- tween the last-named genus and the LEPTOTHRIX. The systematic naturalist, in his attempt to establish genera and species among these lowly organisms, meets with difficulties even greater than those encountered in the classification of the higher cryptogams and flower- ing plants. These difficulties arise from the multitude of species and minute size of the unicellular organisms under consideration ; from the various phases which the same species may present at different epochs in the life-history of the plant; from the morphological identity of species having different physiological char- acters ; and, finally, from the influence of the environ- ment in modifying both morphological and physiological characters. Most writers continue to speak of the Bacteria as fungi. The observations of the writer are, however, in favor of the view of Cohn, that they are more nearly related to the algae. It would be idle, however, to discuss this question, as the border-line of these two great classes of the VEGETABLE KINGDOM is not well defined ; and here, as in the attempt to establish gen- era and species, the systematic naturalist must ever encounter the stubborn fact that NATURE is contin- uous, and, consequently, that all attempts at classifi- cation are artificial. The writer ventures to hope that the resumS given in the present volume will be found to fairly represent the PREFACE. V present state of science as regards the minute organ- isms of which it treats. No doubt the book contains much that will not bear rigid scientific criticism ; and the constant additions to our knowledge which are daily being made will necessitate frequent revisions and additions, if a favorable reception by the Medical Pro- fession, and students of Biology, makes it practicable ?or the writer to carry out his present intention of rep- resenting in future editions the progress which may be uade in the interval. I am not prepared to say, how- ever, that the heliotype plates which illustrate this 3dition will appear in subsequent editions, if they are called for. These plates add greatly to the cost of the volume, and they will perhaps be less satisfactory than lithographs or wood-cuts to those not accustomed to similar views under the microscope, and to those critics who are not familiar with the technical difficulties attending an attempt to photograph the minute organ- isms here represented. If the clean field and sharply- drawn outlines which it is so easy to draw upon wood or stone makes a prettier picture, and one which may be preferred by some, there can be no doubt that these views from nature, if closely studied, are more instruc- tive than drawings, notwithstanding the inevitable de- fects arising in some instances from the presence in the field of view of extraneous objects, and from the im- possibility of having every part of the field in the best possible focus at the same time in these photo-micro- graphs, which are made with objectives of high power having an extremely limited focal range. G. M. S. FORT MASON, SAN FRANCISCO, August 15, 1883. INTRODUCTION. PART FIRST. MORPHOLOGY OF THE BACTERIA, PART SECOND. PHYSIOLOGY OF THE BACTERIA. BY DR. ANTOINE MAGNIN. TRANSLATED FROM THE FRENCH BY DR. GEORGE M. STERNBERG. PEEFACE BY TRANSLATOR. HAVING found the admirable rezumS of our knowl- edge of the bacteria, by Dr. Magnin, of great assistance to me, in pursuing the investigations in which I have been engaged during the past year under the auspices of the National Board of Health, it has seemed to me that a translation of the work into English and its publi- cation in this country would be productive of good in more ways than one, and of the advancement of science. To the naturalist, it cannot fail to be of value, as the most approved classification, that of Cohn, is given, with a full description of species. To give additional value to this portion of the work, figures of many of the best-known forms, drawn from various foreign sources, and reproductions of some of my own photo-micro- graphs (by permission of the National Board of Health), have been introduced. If we are to judge from the scanty literature of the subject in this country, the amount of interest which has been aroused by the revelation of a new world of micro-organisms, and by the momentous questions which have been raised in connection with them, is far below that awakened in Germany, France, and England. This is not, however, really the case ; for, while we have but few active workers in the difficult fields of inquiry X PREFACE BY TRANSLATOR. which have proved so attractive, especially for the Ger- man and the French savants, there is nevertheless a wide-spread interest in these investigations, and a desire to know their results. But, just here, we are met with a difficulty which has no doubt discouraged many, and perhaps caused some to drop the whole subject in dis- gust. The results have been so contradictory, and so many would-be savants have uttered opinions entirely opposed the one to the other, that we find it impossible to arrive at any definite opinion, not knowing whom to believe. This being the condition of affairs, it seems to me that it is necessary for us to commence investigating for ourselves, — first making ourselves familiar with what has been done abroad, and then avoiding, if possible, the quicksands into which unfortunate science has too often been dragged by her votaries. One great trouble which we have experienced in this country is in judg- ing of the comparative value of the observations of dif- ferent men who are equally unknown to us. A very plausible article may be written by a very careless observer ; or a very cautious observer may fail to give confidence in his results, because of a certain degree of confusion in his language. When experiments are well devised, carefully executed, and described with preci- sion, as is done by such men as Pasteur and Tyndall, we cannot fail 'to attach great weight to the conclusions reached. And when so accomplished a microscopist as Cohn or Koch asserts that he has seen such and such a thing, or has made such and such measurements, we cannot doubt the reliability of the observation. But sometimes we are deceived by giving credence to a man who has achieved reputation in one line of study, but of PREFACE BY TRANSLATOR. XI whose skill and training in the use of the microscope we have no means of judging. Such a man may be a great surgeon, or a great clinician, or a great chemist, arid yet be a mere tyro with the microscope. When, then, we see it announced that Dr. So-and-so failed to discover any micrococci in pus, in blood, or what not, taken from a certain source, we are justified in asking, — first, what power did the learned doctor use ? second, is he capa- ble of distinguishing micrococci in fluids which contain them beyond question ? Or, if he does discover them, we may ask if he is accustomed to making a differential diagnosis between micrococci and inorganic granular material, or unorganized granules of organic origin. This is a decision which the most accomplished micro- scopist is sometimes unable to make, except by the aid of chemical tests and culture experiments. To avoid this want of confidence in results, which has naturally grown out of carelessly made observations and contradictory statements, it is desirable that full and minute details should be given of all observations and experiments made, and, whenever possible, that photo- micrographs should be made of all micro-organisms described, or of a thin stratum of a liquid asserted not to contain any ; as, when a sufficiently high power is used, this settles the question of their presence or absence, beyond dispute, and enables other students to make comparisons and measurements which cannot fail to promote the interests of true science. The National Board of Health of the United States has the credit of first adopting this method of recording the results of scientific investigation, in this direction, as a constant and unimpeachable record of what has Xll PREFACE BY TRANSLATOR. been seen by the investigator. The commission sent to Havana last summer for the investigation of yellow fever, was instructed to pursue this method, and was accompanied by a photographer and supplied with all the necessary appliances for carrying these instructions into effect. The superficial reader may find much to criticise in the work of Dr. Magnin, but I am convinced that those who read it carefully cannot fail to be pleased with the truly scientific spirit in which it is written ; the fairness with which conflicting opinions are stated ; the caution manifested as to the drawing of definite conclusions where questions are still under discussion ; and, above all, the extent of his literary researches and the systematic way in which he has arranged the re- sults. For the naturalist, for the physician, or for the non- professional man of general culture, who desires to have accessible in a condensed form the most important re- sults achieved in this line of inquiry up to the present day, this volume cannot fail to be of value ; while for the student and the investigator in search of fuller information, the summary given of the labors of nu- merous individuals, together with the copious bibliog- raphy, which I have brought up to date, will doubtless be of service. Believing this to be true, it has been a pleasure for me to devote a portion of my summer vaca- tion to the translation of this little volume. G. M. S. SALEM, MASS., August 1, 1880. TABLE OF CONTENTS. PAGE INTRODUCTION 11 HISTORICAL 13 PART FIRST. MORPHOLOGY OF THE BACTERIA. CHAPTER I. — ORGANIZATION. § 1. — OF THE BACTERIA IN GENERAL 28 Forms 28 Dimensions 29 Colors 31 Movements 32 Structure 35 Cell-Membrane 35 Protoplasm 36 Cilia 39 § 2. — DIFFERENT MODES OF ASSOCIATION 43 Form of Little Chain (Torula) 43 Form of Zooglcea 44 Form of Mycoderma, &c 45 CHAPTER II. — CLASSIFICATION AND DESCRIPTION. § 1. — PLACE OF THE BACTERIA 48 Among Organized Beings 53 In the Vegetable Kingdom 55 § 2. — CLASSIFICATION 59 Characters Generic and Specific 60 Classification of Cohn . 65 XIV TABLE OF CONTENTS. PAGE § 3. — DESCRIPTION OF GENERA AND SPECIES .... 65 Spherobacteria 71 Micrococcus 72 Monads 78 Microbacteria 80 Bacterium 80 Desmobacteria 88 Bacillus 87 Leptotlirix 90 Spirobacteria 91 Vibrio 92 Spirillum 94 PART SECOND. PHYSIOLOGY OF THE BACTERIA. CHAPTER I — DEVELOPMENT IN GENERAL. § 1. — ORIGIN OF BACTERIA 101 Heterogenesis 102 Dissemination 103 In Air 103 In Water 106 In the Human Organism 107 § 2. — NUTRITION AND RESPIRATION Ill Aliments : Water Ill Nitrogen 112 Carbon 113 Oxygen 115 Temperature 118 Other Agents 121 § 3. — REPRODUCTION 123 Fission 123 Spores 126 Sporangia 130 Polymorphism 133 TABLE OF CONTENTS. XV CHAPTER II. — DEVELOPMENT IN DIFFERENT MEDIA. PAGE £1. — ROLE OF BACTERIA IN FERMENTATIONS .... 137 Acetic Fermentation 139 Ammoniacal Fermentation . 142 Lactic and Butyric Fermentation 144 § 2. — ROLE IN PUTREFACTION AND NITRIFICATION . . 148 PART THIRD. TECHNOLOGY. § 1. — METHODS OF CULTIVATION 156 Methods of obtaining Pure Stock 156 Natural Culture-Fluids 161 Blood 161 Milk 163 Urine 164 Aqueous Humor 165 Artificial Culture-Fluids 167 Sterilization of Culture-Fluids 168 Culture Tubes and Flasks 171 Culture Flasks used by the Author 175 Culture Oven 180 Thermostat for Gas 181 § 2. — THE RECOGNITION OF BACTERIA 184 § 3. — STAINING BACTERIA 186 Staining the Tubercle Bacillus 190 Baumgarten's Method 191 Ehrlich's Method 191 Gibbs' Method 192 § 4. — PHOTOGRAPHING BACTERIA 194 § 5. — COLLECTION OF ATMOSPHERIC BACTERIA .... 197 Examination of Water 201 § 6. — ATTENUATION OF VIRUS 201 Method of Pasteur . 202 XVI TABLE OF CONTENTS. PAGE Method of Toussaint 204 Method of Chauveau 205 Method by Intravenous Injection 206* Attenuation of Virus by Chemical Reagents . . 206 PART FOURTH. GEKMICIDES AND ANTISEPTICS. Acetic Acid 215 Alcohol 215 Aluminium Acetate 216 Aluminium Chloride 216 Ammonia 216 Aromatic Products of Decomposition . . 216 Arsenious Acid 216 Benzoic Acid 217 Boric Acid 217 Bromine 218 Camphor 218 Carbonic Acid 218 Carbonic Oxide 219 Carbolic Acid 219 Chloral Hydrate 220 Chloroform 220 Chlorine 220 Chromic Acid 221 Citric Acid 221 Creosote 222 Cupric Sulphate 222 Ether 222 Eucalyptol 222 Ferric Sulphate 222 Ferri Chloridi Tinct 223 Glycerine 223 Heat 223 Hydrochloric Acid 224 Hydrogen 225 Iodine 225 Mercuric Bichloride , . 225 TABLE OF CONTENTS. XVli PAGE Nitric Acid ' 226 Xitrous Acid 226 Oil of Mustard . . 226 Oil of Turpentine 226 Osmic Acid 226 Oxalic Acid 226 Ozone 227 Oxygen 227 Picric Acid 227 Potash 227 Potassium Arsenite • 228 Potassium Chlorate 228 Potassium Iodide 228 Potassium Nitrate 228 Potassium Permanganate 228 Pyrogallic Acid 229 Pyroligneous Acid 229 Quinine 229 Salicylic Acid 229 Soda 230 Sodium Biborate 231 Sodium Chloride 231 Sodium Hyposulphite 232 Sodium Sulphite 232 Sodium Salicylate 232 Sulphuric Acid . . . . . * . . . 233 Sulphurous Acid 233 Sulphuretted Hydrogen «... 234 Tannic Acid 234 Thymol 234 Zinc Chloride 234 Zinc Sulphate 235 PART FIFTH. BACTERIA IN INFECTIOUS DISEASES. Anthrax 265 •Symptomatic Anthrax 280 Cerebro- Spinal Meningitis . . . , 284 XV111 TABLE OF CONTENTS. PAGE Cholera 285 Erysipelas 286 Cholera of Fowls 288 Diphtheria 291 Disease produced by Bacilli 297 Fatal Epidemic among Fish caused by Bacteria 299 Glanders 29!) Gonorrhoea 301 Hydrophobia 314 Intermittent Fever 317 Leprosy 331 Malignant (Edema 336 Milk Sickness 339 Measles 340 Pleuro-Fneumonia 341 Infectious Pneumonia 312 Pyannia in Rabbits 31-3 Relapsing- Fever 346 Scarlet Fever 349 Septicaemia in Mice 351 Septicaemia in Rabbits . . . 355 Spreading Abscess in Rabbits 376 Swine Plague 378 Syphilis 3*0 Tuberculosis 384 Typhoid Fever j. 400 Ulcerative Endocarditis 411 Variola 411 Variola of Pigeons 413 Whooping Cough 41") Yellow Fever . . 417 PART SIXTH. BACTERIA IN SURGICAL LESIONS . '442 BIBLIOGRAPHY 457 INDEX 489 LIST OF PLATES, PLATE PAGE I. The Cilia of B. termo and S. voluntans. (Drysdale and Dallinger) 40 II. Different Modes of Grouping of the Bacteria. (Photo- micrographs by Dr. Sternberg) 47 III. Saccharomycetes and Schizomycetes. (Photo-micro- graphs by Dr. Sternberg) 58 IV. Disease-Ferments of Wort and Beer. (Pasteur) . . 84 V. Different Forms of Bacteria. (Cohn) 95 VI. Different Forms of Bacteria. (Photo-micrographs by Dr. Sternberg) 98 VII. Dissemination of the Bacteria. (Photo-micrographs by Dr. Sternberg)' 100 VIII. Reproduction of Bacillus Ulna, by Spores. (Photo- micrographs by Dr. Sternberg) 127 IX. Micrococci. (Photo-micrographs by Dr. Sternberg) 154 X. Spirillum Obermeieri and B'acillus antliracis. (Koch) . 264 XI. Bacilli. (Photo-micrographs by Dr. Sternberg) . . 272 XII. Blood of Yellow Fever Patients. (Photo-micrographs by Dr. Sternberg) 424 THE BACTERIA. INTRODUCTION. " Corruptio unius est generatio alterius." LUCRETIUS, De Rerun, Datura. OF all the studies which have for their object the inferior organisms, those which relate to the bacteria offer, without contradiction, the greatest interest, as they touch the most diverse problems, which, it is true, are the most difficult and the least known in biology. The history of these mi- nute organisms is, in truth, related to that of spontaneous generation, to that of the fermenta- tions, to the pathogeny and therapeutics of a great number of virulent and contagious affections, and, in a more general manner, to all the .unknown which, notwithstanding the efforts of modern sci- ence, still surrounds the origin of life and its pres- ervation. If the relation of these inferior organisms to the origin of living beings is yet obscure, their role in the preservation of life is better known. It is known that organic matter, once produced and become solid, so to speak, cannot again enter into the general current until it has undergone 12 THE BACTERIA. new transformations, metamorphoses produced, ac- cording to some savants, favored, according to others, but, without contradiction, accompanied by the development of bacteria ; 1 and, without wishing to attribute to these organisms a finality which is repugnant to our monistic conception of the universe, it may be said that it is thanks to them that the continuation of life is possible on the surface of the globe. But, if these studies are full of interest, their field is so vast that we cannot flatter ourselves that we have passed over the whole of it with equal care. The little time that has been ac- corded us for the composition of this thesis will be our excuse for the inevitable imperfections which will doubtless be found in our work. 1 The bacteria: such is the subject which has been imposed upon us; but it is certainly useless to give the reasons which have caused us to study not only the bacteria properly so calk-d, taking the word in its most restricted sense, but all the organisms which are comprised under the names of bacteria, vibrios, schizomyeetes, schizophytes, etc. HISTORICAL. THE bacteria are the lowest organisms, situated upon the limit of the two kingdoms, animal and vegetable, and are thus defined by the botanists who have most recently occupied themselves with them : — " Cells deprived of chlorophyll, of globular, ob- long, or cylindrical form, sometimes sinuous and twisted, reproducing themselves exclusively by transverse division,1 living isolated or in cellular families, and having affinities which approach them to the algae and especially to the oscillatoriae." But, before arriving at this degree of relative precision, the history of the bacteria has passed through the most diverse vicissitudes. At one time considered as animals, at another taken for vegetables, transported from the algae to the fungi, one author has even gone so far as to refuse to them the nature of living beings.2 This diversity of opinions is due to the minuteness of* their di- mensions and the difficulties with wrhich their ob- servation is surrounded. 1 Keprod action by spores has been proved to occur in Bacillus subtil is, and it seems altogether probable that other species are reproduced in the same way. — G. M. S. 2 Polotebnow. 14 THE BACTERIA. Although an historical statement of the progress of our knowledge of these minute organisms has been given in several publications, we think it best to make here a new historical summary, which will be completed by an indication of the principal pa- pers relating to them which have been published recently. The first observer who perceived bacteria was Leeuwenhoeck. As early as 1675, while examin- ing by chance with his magnifying glasses a drop of putrid water, the father of microscopy re- marked with profound astonishment that it con- tained a multitude of little globules, which moved with agility. The following year he recognized the presence of bacteria in fasces and in tartar from the teeth ; and, if he has not named them, it is easy to assure one's self by the description which he has given of their form and of their movements, and by the figures which accompany these descriptions,1 that the organisms observed by him are truly Bacteria, Vibrios, and perhaps even Leptothrix. In 1773 0. F. Miiller endeavored to classify these organisms. He made of them a group of infusoria, under the name of Infusoria crassius- cula, and^ established two genera, — the g. Monas and Vibrio ; the first characterized as follows : " vermis inconspicuus, simplicissimus, pellucidus, punctiformis," comprising the following species: Monas termo, atomics, pundum, ocellus, lens, mica, 1 Leeuwenhoeck. Opera omnia, Lugd. Batar., 1722, 11, p. 40, fig. A to G. HISTORICAL. 15 tranquilla, lamellula, pulvisculus, uva, which it is impossible to identify with the species at pres- ent recognized. The genus Vibrio " vermis incon- spicuus, simplicissimus, teres, elongatus" enclosing under thirty-five specific names, with the true bacteria, some organisms belonging to other classes of the animal and vegetable kingdoms. In the classification of - the infusoria given by Bory de Saint-Vincent in the " Encyclopedic Methodique " (1824) and afterwards in the "Dic- tionaire Classique d'Histoire Naturelle " (1830) the bacteria are distributed in two different families of the microscopic gymnodae, the monadaires and the vibrionides. Besides the monads, properly so called, of which the Monas termo has been pre- served by the greater part of the bacterologists, the monadaires include some veritable infusoria, which have no relation with the monads. It was the same with the vibrionides, of which the genera Vibrio and Mellanella included some beings very different in their organization. Indeed, beside some veritable vibrios, bacteria, and spirilla, constituting the genus Mellanella, Bory placed some nematoid worms, such as the Anguillula of vinegar. With Ehrenberg (1838) and Dujardin (1841) the family of the vibrioniens was established upon characters more homogeneous, and their species upon distinctions truly scientific. But these two observers, followed in this by M. Davaine, deny completely the affinities of the elongated bacteria 16 THE BACTERIA. (Bacterium, Vibrio, etc.) with the punctiform bacteria (Monas) ; and it is necessary to come to the time of MM. Hallier, Hoffmann, Cohn, arid the greater number of recent botanists, in order to see these two forms brought together anew. In fact, Ehrenberg defines his vibrioniens, which he arranges between the volvocineae and the closteria " animals, filiform, distinctly or appar- ently polygastric, no mucous membrane, naked, without external organs, with the body (like mon- ads) uniform and united in chains or filiform se- ries, as a result of incomplete division." He included in this class all filiform bodies gifted with proper movement and formed of articles, dividing them into four genera : — 1. Bacterium: filaments linear and inflexible; three species. 2. Vibrio : filaments linear, snakelike, flexible ; nine species. 3. Spirillum : filaments spiral, inflexible ; three spe- cies. 4. Spirochcete : filaments spiral, flexible; one species. A fifth genus, including but one species, the Spirodiscus fulvus, with filaments in a helix, in- flexible, disposed in contiguous layers, has not been seen since Ehrenberg. Let us add that Ehrenberg often attributed to them a complex structure, stomachs more or less numerous, a pro- boscis, cilia serving as organs of locomotion, — • all characters that more recent observers have failed to find. Nevertheless, we must make an HISTORICAL. . 17 exception in favor of the cilia, of which the ex- istence has been recently verified in the case of several of the bacteria by divers botanists, among others by MM. Colm and Eug. Warming. Dujardin (1841), in his " Histoire Naturelle des Zoophytes," preserved the family of the vibrioni- ens of Ehrenberg among the infusoria, characteriz- ing them as follows: "filiform animals, extremely slender, without appreciable organization, without visible locomotive organs." He made but few modifications, of which the principal consisted in uniting Spirochceta with Spirillum, Dujardin. Re- jecting the character that Ehrenberg drew from the rigidity of the spirilla, the Spirochceta plica- tilis, Ehrb. became the Spirillum plwaiile, Duj. ; but, as will be seen later, this change has not been maintained. Dujardin, then, classed the bac- teria in : 1. Bacterium : filaments rigid, with a vacillating movement. 2. Vibrio : filaments flexible, with an undulatory movement. 3. Spirillum : filaments spiral, movement rotatory. Until this time the bacteria had been considered as animals placed at the foot of the series. Sub- sequently the tendency to place them in the vegetable kingdom became more and more pro- nounced. Already, since 1853, M. Ch. Robin had pointed out the relationship of the bacteria and of the 18 THE BACTERIA. vibrios with Leptoihrix. This opinion, which was not favorably received by the authors who adopted nearly all of the generic groups of Ehren- berg and Dujardin, is to-day accepted by many botanists, above all since the labors of Cohn. (See below: classification.) At all events, it is to M. Davaine (1859) that we are indebted for clearly pointing out that the vibrioniens are vegetables, nearly allied to the algse, and especially to the confervae. This same author, having observed some mo- tionless bacteria, thought it necessary to give this character great consideration, and to estab- lish a fourth group, the genus Bacteridium, which he added to the three others admitted by Dujar- din ; but in this creation he was less happy than in his placing the vibrioniens among the vege- tables ; for we shall see further on that this char- acter of mobility or of immobility is not absolute, and that it depends upon the age of the bacterium or upon certain conditions relating to the medium in which it is placed. The most recent complete exposition of the classification and of the ideas of M. Davaine is found in the " Dictionnaire Encyclop. des Sci- ences Medicales," art. Bacteries (1868). It may be summed up as follows : — Filaments straight or bent, but not in a spiral Moving sponta-) Rigid. . BACTERIUM. neously . . j Flexible. VIBRIO. Motionless BACTERIDIUM. Filaments spiral SPIRILLUM. HISTORICAL. 19 The genus Bacterium comprises six species, — B. termo, catenula, punctum, triloculare, or articula- tum, already described by Ehrenberg and Dujar- din, and B. putredinis and capitatum, new species of M. Davaine, established, the first for a bacte- rium producing rot in plants, the second for a spe- cies, swollen at the extremity, observed in some macerations. The genus Vibrio includes twelve species, — V. lineola, tremulans, rugula, prolifer, serpens, bacillus, synxanthus, and syncyanus of previous authors and the V. lactic, butyric, and tartaric right, discovered by M. Pasteur in these different fermentations. In the genus Bacteridium, M. Davaine places five new species, — the " Bacteridies charbonneuse, intestinale, du levain, glair euse, et des infusions." He includes also the ferment which, according to M. Pasteur, occasions the " sickness of turned wine." Finally, the genus Spirillum includes the spe- cies S. undula, tenue, volutans of Ehrenberg, 8. rufum and leucomcenum of Perty, and 8. plicatile, Duj. From this moment the history of the bacteria enters upon a new phase. The labors of M. Pas- teur upon the inferior organisms and their role in fermentation, the researches of MM. Davaine and Hallier upon the bacterium of charbon, and the micrococci of contagious maladies, call the atten- tion of chemists and of pathologists to these or- 20 THE BACTERIA. ganisms and especially to the bacteria. Their origin, their evolution, the physiological peculi- arities of their nutrition and reproduction, are the object of numerous labors, and .give rise to passionate discussions relating to the subject of spontaneous generation, polymorphism of fungi, theories of fermentation, and the pathology of virulent and infectious maladies. For this reason an exposition of these researches, often contradic- tory, is extremely difficult. We will make it suc- cinctly, insisting especially upon the labors relating to the classification of the bacteria, and reserving to ourselves the privilege of returning to the his- tory of several points, when we approach their study in the special chapters of this thesis. The first important memoir published after that of M. Davaine upon the bacteria is that of M. Hoffmann, in 1869. He demonstrates: First, that the bacteria are plants, having a very distinct cellular organization ; second, that they can only be classified in accordance with their form and size, at first into monads and linear bacteria, and the latter into microbacteria, mesobacteria, and megabacteria ; (M. Hoffmann includes with the linear bacteria, Vibrio, Bacterium, and LeptothriXj which are bacteria united in a chaplet ;) third, that mobility or immobility is not a specific char- acter, but may present itself in the same species under the influence of changes of temperature, of density of medium, etc. M. Hoffmann studies also the origin of the bacteria, and rejects the hypothesis of a spontaneous generation. As to HISTORICAL. • 21 their role in the phenomena of the decomposition of organic bodies and in fermentations, M. Hoff- mann confesses " that, with the exception of yeast and of the acetic and butyric ferments, all the rest is still enveloped in obscurity." M. Cohn is the naturalist who, in our days, has occupied himself the most with the bacteria. In 1853, he published his first researches upon this subject. The genera Zooglcea, which he estab- lished at this time for the bacteria arranged in ge- latinous masses, diffused or more or less crowded together, was not a happy creation. It was adopt- ed at first by M. Rabenhorst who, in his work on the fresh-water algae of Europe, places them after the palmellaceae, while he classes the other bac- teria, Vibrio and Spirillum, in the family of the oscillatoriae. The Zoogloea are later abandoned by their author as a generic group, and are preserved only as the name of one of the diverse transitory stages through which the bacteria pass in the course of their evolution (Zooglcea, Leptothrix, Torula). Twenty years later the same savant commenced the publication of a series of " Memoirs" upon these organisms (in his " Beitrage zur Biologie der Pflanzen"). In the first paper the author gives an exposition of his researches upon the organization, development, and classification of the bacteria, and upon their action as ferments. M. Cohn considers them as a well-defined group, — the schizospores, belonging to the algae, at the commencement of the series of the phycochroma- 22 THE BACTERIA. cese, with several families with which the different genera of bacteria have many affinities. He rec- ognized, however, that the absence of chlorophyll approaches them, at least from a functional point of view, to the fungi. Upon this point we may say that for other botanists this character is de- cisive, and the bacteria are classed as fungi. M. Nageli, who takes this view, describes them under the name of Schlzomycetes. Cohn divides the bacteria into four tribes, comprising six genera : — 1. The Sphcerolacteria or globular B. 2. The Microbacteria or rod B. 3. The Desmobacteria or filamentous B. 4. The Spirobacteria or Spiral B. We will return to this classification. In 1874, M. Th. Billroth, in his researches upon the Coccobacteria septica, expressed opinions en- tirely different from those of Cohn. According to Billroth, the bacteria differ considerably in form according to the medium in which they are placed and divers circumstances. He claims that they constitute but a single species, the Coccobac- teria septica. This vegetable organism can pre- sent itself under the form of globular articles (coccos) or under that of rods (bacterie). These two forms may reproduce themselves by becoming elongated and dividing transversely, or may pass the one into the other. Billroth claims to have found both forms united in a single filament, a HISTORICAL. 23 fact which in his opinion demonstrates conclu- sively their relationship. Each of these two forms can also present variations of size, in ac- cordance with which he establishes the following divisions : — Micrococcos Microbacteria. Mesococcos Mesobacteria. Megacoccos Megabacteria. And varieties of association which give rise to the following names : — Monococcos Monobacteria. Diplococcos Diplobacteria. Streptococcos Strep tobacteria. Gliacoccos Gliabacteria. Petalococcos Petalobacteria. Ascoccos. The following year (1875), Cohn, in the second part of his " Researches " upon the bacteria, criti- cised the opinions expressed by Billroth in the pre- ceding memoir. Cohn believes that we should regard as distinct genera and species ail the bac- teria having a particular form and acting differ- ently as ferments, so long as the proof of their identity has not been demonstrated in an evident manner. Coming back also to the affinities and classification of these organisms, he insists anew upon their near relationship to the Phycochro- macese; and, no longer distinguishing the bac- teria as a special family, he distributes his different genera in a group, which he calls Schi- zo2)hytes, which includes the greater part of the 24 THE BACTERIA. Chrococcece and of the Oscillarice. We will re- turn to this subject when we speak of the clas- sification of the bacteria. In 1876, appeared in the same number of Cohn's " Beitrage " two important papers. The first, by Cohn, treats of the influence of tempera- ture upon the bacteria, of their origin, of the formation of spores in the Bacillus of hay infu- sion, and of charbon. The second, by Koch, gives the result of his researches upon the bac- teria of charbon, the Bacillus anthracis. Koch has been able by skilful cultivation to follow the complete development of this Bacillus, and to witness the formation of spores, of which the vitality is very great, and which are the prin- cipal agents of the transmission of this terrible malady. I must still indicate, in addition to these special works, a quantity of notes and of memoirs scat- tered through the reviews and periodical publica- tions. The list will be found in the bibliography ap- pended to this work. I must also cite the recent work of M. Nageli upon " The Inferior Fungi and their Role in Infectious Maladies." The learned professor of Munich has studied the di- verse fungi which produce decompositions. He divides them into three groups, — the Mucorini, the Saccharomycetes, and the ScAisomycetes, which correspond to the bacteria. According to Nageli, HISTORICAL. 25 the bacteria are fungi which produce putrefac- tion. In presence of these opinions, so diverse, as to the nature of the bacteria and their classification, we will finish by saying with Cohn : — " So long as the makers of microscopes do not place at our disposal much higher powers, and, as far as possible, without immersion, we will find ourselves, in the domain of the bacteria, in the situation of a traveller who wanders in an un- known country at the hour of twilight, at the moment when the Mght of day no longer suffices to enable him clearly to distinguish objects, and when he is conscious that, notwithstanding all his precautions, he is liable to lose his way." PART FIRST. MOEPHOLOGY OF THE BACTERIA. CHAPTER I. ORGANIZATION OF THE BACTERIA. WHEN bacteria develop in a liquid in a suffi- cient quantity, they become visible to the naked eye. They appear either as a slight cloud, or gathered in little masses in the liquid, or forming a pellicle upon its surface, or as a deposit upon the walls of the vessel and upon the objects contained in the liquid. However, we must hasten to say with M. Cohn, that the fact of the absence of all turbidity in a liquid does not exclude the possi- bility of the presence of bacteria. In liquids more dense than water (serum, lymph, etc.), when the refractive power of these corpuscles is the same as that of the liquid, their presence may not be revealed to the naked eye. We will add that sometimes their color serves to indicate their presence in a liquid, although this color is often very feeble, and can only be perceived when a considerable thickness of the liquid is examined. If we examine these clouds, these accumulations, these deposits, with the microscope, we see that 28 MORPHOLOGY OF THE BACTERIA. they are formed of a myriad of little bodies iso- lated or grouped, globular or linear, gifted or not with motion, sometimes colored. These variations constitute so many characters which require to be studied with some detail. § 1. BACTERIA IN GENERAL. Form. — The bacteria, as understood to-day by most botanists, when considered in their separate state, are of two principal forms, — globular bod- ies, or monads, and bodies more or less filiform, or bacteria properly so called. The globular bacteria comprise organisms round- ed, ovoid, sometimes elongating themselves into a tube (Warming). The Monas crepusculum of Ehrenberg may be taken as a type. This form includes also the Micrococcus of Hallier, the Mi- crosporon of Klebs, the round forms of the Amy- lobacter of M. Trecul, and perhaps the Microzyma of M. Be champ. We will see farther on that these are very probably phases of development of the spores of bacteria, properly so called. The bacteria, not globular, present a greater diversity of form ; they may be straight, undu- lating, or twisted in a spiral. The rectilinear bacteria are usually exactly cylindrical throughout their whole extent ; and in this case they form very short cylinders, as in the Bacterium., Cohn, or cylinders of which the length is several times as great as the thickness, as in the Bacteridies (Bacillus ulna Cohn) ; others are ORGANIZATION OF THE BACTERIA. 29 swollen in the middle, with their extremities rounded, such as certain forms of Vibrio serpens (Warming) ; others again are fusiform, swollen in the middle and attenuated at the extremities, — Bacterium fusiforme (Warming) ; rectilinear bac- teria swollen at the two extremities are met during the life of certain species, B. lineola and B. termo, for example, above all when they are transported to a more favorable medium : this modification usually precedes segmentation ; final- ly, one meets sometimes bacteria swollen at one extremity only; the swollen part presents often a clear point and sometimes an evident spore : we shall see later the signification of this peculiarity. With these claviform bacteria we may include the Bacterium capitatum Dav., the Helobacteria of Billroth, and certain Amylobacter, with heads of the Ficus carica, etc. (Ch. Robin). The undulating bacteria constitute the Vibrios properly so called (V. rugula, serpens, etc.). The spiral bacteria of which the turns are more or less elongated are named Spirillum, Spiro- chceta, etc. Dimensions. — The dimensions of the bacteria oscillate between the most variable limits, but in a general way it may be said that they are the smallest of all microscopic beings. Some of them are situated at the extreme limit of our highest magnifying powers ; and their proportions, as to 30 MORPHOLOGY OF THE BACTERIA. length and thickness, are comprised within the limits of errors of observation. The globular bacteria are the smallest, and the dimensions of some species are so minute that they cannot be measured directly. The largest are the Spirillum, which attain a length of T27 of a millimetre. Between these two extremes, there are all intermediary sizes possible. The dimensions of some of the bacteria are given below: — Monas vinosa, 0.5 to 1 /-t, in diameter; length 3 to 4 fjb. Bacterium termo, breadth 0.6 to 0.8 //,; length 2 to 3 p. Vibrio lineola, breadth 0.5 to 1 p ; length 3 to 8 p. Bacillus ulna, „ 0.7 to 1 p ; „ 5 to 8 /u,. B. anthracis, „ 1 to 2 p ; „ 10 to 50 p. Spirillumvolutans, „ 7 /j, ; „ 10 to 40 ^ Several authors, considering exclusively this character of dimensions, have divided the monera and the bacteria according to their size. Thus Hoffmann recognizes in addition to the monera, only the microbacteria, the mesobacteria, and the macrobacteria. In the same way Billroth classi- fies the monads according to their dimensions into micro, meso, mega coccos, and the bacteria into micro, meso, mega bacteria. Finally, Klebs sep- arates the Micrococcos from the Microsporines, which do not differ from them except by their smaller dimensions, both forms being able to pass to the state of bacteria (rods). ORGANIZATION OF THE BACTERIA. 31 Color. — The phenomena relating to the color of bacteria have only recently been pointed out. " But little attention has been given to the color of the bacteria, regarded generally as colorless," said M. de Seynes in 1874 ; and recently M. de Lanessan, " The bacteria are ordinarily quite color- less." However, M. Cohn had already insisted upon the globular bacteria chromogenes, or of pig- mentary fermentation, and upon the colors pro- duced ty different monads, which have long since been studied by microscopists. Upon this subject, let us observe that the bac- teria wlich are colored belong to two very dif- ferent groups. First, colored organisms always known a^ such, but which were not formerly in- cluded wi\}i the bacteria, as the different monads, which ha^ become the Micrococcus prodigiosus, cyaneus, awantiacus,Cohn, etc.; the second group includes thV bacteria properly so called, which absorb the Coloring matter of vegetables upon which they a» fixed as parasites, or of the media in which they\|ive. This is the case with the bac- teria observed W M. de Seynes upon the Penicil- lium glaucum, aid perhaps with the Vibrio sijn- xanthus and s?/^am^?Ehrenb., which give to milk a yellow or blueVolor according to the species. We will return toVhis subject when we speak of the nutrition of th\ bacteria. As to the purplVcolored monads, they have been especially studied as early as 1838 by Dunal, then by Morren and Yhrenberg, and in our own day by Kay-Lankester\Cohn, Klein, and finally o2 MORPHOLOGY OF THE BACTERIA/ by Warming and Giard. They are found in va- rious media — in sea- water, in hot sulphur springs, in fresh water containing animal or vegetable mat- ter in a state of putrefaction. They appear some- times upon bread, meats, and in general upon cooked food placed in a humid atmosphere. The different colors which they present are red, yel- low, orange, and blue. It is probably to anal- ogous organisms that we must attribute tie blue color presented by pus under certain circum- stances, the green and blue color studied by M. Chalvet, and the orange-yellow, bright red, and blue colors observed by C. Eberth h perspi- ration. In Norway, red bacteria appear in simmer in such masses that the borders of the sea are some- times colored of an intense red (Warning). Movement. — The bacteria are met in two dif- ferent states. They are active or moionless ; but it is now well settled for the greater number that the same species may present itseF sometimes in a state of repose, sometimes in a state of move- ment. The movements of the bacteria are of two kinds, — a movement of the corpuscle upon itself and a movement of translation. Th( first is sometimes nothing more than a molecula or brownien move- ment, which occurs in the srrtHest forms. But at other times it is more exte-ded, and consists in a movement of rotation rou:4 the axis, or a bend- ing of the body. This flexibility is, above all, ORGANIZATION OF THE BACTERIA. 33 observed in the long forms, the Bacillus, the Vib- rions, etc. As to the movement of translation, it is very variable ; at one time slow, at another rapid, it is in relation with the length and form of the bacterium. M. Cohn has well described all the modifications of movement in the following lines : — " Almost all the bacteria possess two different modes of life, characterized by repose and by movement. "In certain conditions, they are excessively mobile ; and when they swarm in a drop of water, they present an attractive spectacle, sim- ilar to that of a swarm of gnats, or an ant-hill. The bacteria advance, swimming, then retreat without turning about, or even describe circular lines. At one time they advance with the ra- pidity of an arrow, at another, they turn upon themselves like a top ; sometimes they remain motionless for a long time, and then dart off like a flash. The long rod-bacteria twist their bodies in swimming, sometimes slowly, sometimes with address and agility, as if they tried to force for themselves a passage through obstacles. It is thus that the fish seeks its way through aquatic plants. They remain sometimes quiet, as if to re- pose an instant: suddenly the little rod commences to oscillate, and then to swim briskly backwards, to again throw itself forward some instants after. All of these movements are accompanied by a second movement analogous to that of a screw which moves in a nut. When the vibrios in the 3 34 MORPHOLOGY OF THE BACTERIA. shape of a gimlet turn rapidly round their axis, they produce a singular illusion : one would be- lieve that they twisted like an eel, although they are extremely rigid." The causes of these movements have been sought, at first, in the supposed animal nature of the bac- teria, and the movements assimilated, consequently, to voluntary movements ; but this opinion can no longer be sustained, as similar movements are to be seen in a great number of vegetable organisms, such as the diatoms, the oscillatoriae, the spores of algae and some fungi, etc. They have also been attributed to the existence of locomotor appen- dices (Ehrenberg) ; but, although the cilia, denied at first by most microscopists, have been seen since in nearly all the bacteria, the botanists who have best studied them, M. Warming, for example, rec- ognize that it is scarcely probable that these or- gans are the cause of their movements, for " one meets some examples in which the body remains motionless while the cilia are in violent agitation, and others in which the body moves while the cilia remain inert, or dragging behind." The movements appear to depend rather upon the nutrition, or respiration, and especially upon the presence of oxygen (Cohn) ; indeed when this gas is wanting the bacteria become motionless. Immobility may also be produced by want of nutriment, poisoning by different toxic substances, (chloroform, iodine, etc.), dessication, etc. The attempt has been made to use the charac- ters derived from the existence or absence of ORGANIZATION OF THE BACTERIA. 35 motion, and the form of the bacteria, in order to classify them ; but what has just been said shows clearly that these transitory phenomena cannot be taken for generic or specific characters. Structure. — It was for a long time believed that the bacteria were constituted of amorphous masses of protoplasm, or of solid rods. The researches of Hoffmann have shown that they have a true cellu- lar structure. We shall describe, then, succes- sively, their membrane, the contents, and the cilia, which may be considered as belonging to the pro- toplasm. Cell-membrane. — The extreme minuteness of the bacteria usually prevents a direct demonstra- tion of the cell-membrane, and the existence of this envelope has not, heretofore, been clearly demonstrated except by indirect proofs ; chemical reactions, for example. Thus Hoffmann verifies the existence of a cellu- lar envelope when " the contents, which is a trans- parent plasma, are partly coagulated, as sometimes happens, or disappear, and are then replaced by air which shows precisely the form of the normal bacterian cell." Warming, also, has not been able to see the membrane, " which only appears dis- tinctly when a vacuole has formed just against the periphery." On the other hand, the action of chemical agents upon bacteria proves that they have an envelope of cellulose, which is colored by tincture of iodine ; 36 MORPHOLOGY OF THE BACTERIA. is not destroyed by caustic potash, ammonia, or even acids; and resists putrefaction for an ex- ceedingly long time. In tins respect, it resem- bles the membrane of cellulose of vegetable cells (Colin). We should add that Cohn claims to have suc- ceeded with high powers in seeing directly the cell-membrane. On the other hand, Warming has never succeeded in so doing. The last observer remarks also that the resistance of bacteria to acids, to alkalis, etc., does not seem to prove the existence of a membrane, " inasmuch as this may be the result of a particular condition of the plasma, which in all the bacteria is of a more con- sistent nature than in other plants." Finally, the membrane may be, in certain bac- teria, tender, flexible and susceptible of move- ments of torsion. In others, it is rigid and incapable of bending. Cohn thinks also that it may swell and dissolve into mucilage, a fact which would explain the origin of this substance in the Zooglcea. Protoplasm. — The contents of the cell is a nitrogenous substance, generally colorless, more highly refractive than water. In the smallest species, this protoplasm appears homogeneous ; but in the bacteria of medium size, and above all in the large species, the contents of the cell encloses portions more highly refractive, vacuoles, special granules, and sometimes diverse coloring matters. ORGANIZATION OF THE BACTERIA. 37 Cohn has first pointed out the movements of the protoplasm, in which currents occur, above all in the central portion, the peripheral portion remain- ing homogeneous and motionless. The vacuoles are also found in the central portion ; "Warming, however, who has observed them in Monas Okenii, Vibrio rugula, V. serpens and Spirillum undula var. littoreum, has sometimes seen them in the mid- dle of the plasma, jit another near the exterior wall. The granules which are seen in the protoplasm were considered by Ehrenberg as stomachal vesi- cles or ovules. They are of two sorts ; the one, highly refractive and not bordered by a dark-circle, are considered by Warming as nothing more than mere compact masses of protoplasm ; the second, also highly refractive, but surrounded by a dark circle, resemble drops of oil, and have been taken for fat granules ; but the recent researches of Cramer, Cohn, and Warming have proved that some of them, at least, are formed of crystalline sulphur. They are not soluble either in hydro- chloric acid or in water, but they are dissolved in absolute alcohol, in hot caustic potash and sulphite of soda, in nitric acid and chlorate of potash at ordinary temperatures, and in bisulphide of carbon, when the membrane, which is permeable with dif- ficulty, has been previously destroyed by sulphuric acid. Although their small dimensions and great refractive power prevent them from being dis- tinguished with certainty as crystals of sulphur, as they are doubly refractive to polarized light their crystalline nature cannot be doubted. MORPHOLOGY OF THE BACTERIA. These globules of sulphur have been observed in Monas Okenii, Bacterium sulphuratum, Oplii- domonas, and the different species of Beggiatoa, both in fresh water, in putrid sea-water, and in thermal sulphur waters. It will be seen when we speak of the physiology of these organisms what their role is in the elimination of sulphur and the formation of sulphuretted hydrogen. We have said, in speaking of the colored bac- teria, that some borrow their color from the sur- rounding medium, and that others, on the contrary, have a color of their own. The protoplasm of the . latter .contains a granular coloring matter, which is ordinarily yellow, blue, or red. The red color- ing matter is most common, and this has been best studied, and appears to be the best known. One of these colors which gives a pink tint (peach color) to Bacterium rubescens, Ray-Lank. ( Clathrocystis roseopersicina, Cohn) ; Monas vinosa, Ehrb., M. Okenii, Cohn; M. gracilis, Warming; Hhabdomonas rosea, Cohn; M. Warmingii, Cohn; Ophidomonas sanguinea, Ehrb. ; Merismopedia littoralis, Rabenh. ; etc., has been studied by Ray- Lankaster, who has given to it the name of bac- terio-purpurine. It is insoluble in water, soluble in alcohol, ether, carbolic acid, glycerine, and fatty oils, — characteristics which make it resemble chlorophyll. It has also a characteristic spectrum. Other red coloring matters which appear to be different have been found in Monas prodigiosa, Ehrb.; Bacillus niber, Cohn; and Micrococcus ful- vus, Cohn. These should not be confounded with ORGANIZATION OF .THE BACTERIA. 39 the purple coloring matter of other algse, as that of the Porphyridium cruentum, which comes from a mixture of chlorophyll and of phycoerythrine. The bacteria never contain chlorophyll. In this connection, it is interesting to recall the protoplasmic constitution of the Amylobacter of Tre'cul. These organisms are, according to Van Tieghem, bacteria, to which he has given the name of Bacillus Amylobacter, and which does not dif- fer from B. subtilis, except by a specific character, extremely transitory, — the presence of amorphous starch, formed and stored in reserve during the period of growth, to be again used later, and con- sumed during the process of reproduction. Cilia. — These appendages which were described by Ehrenberg in the Bacterium trilocular have not been seen since. To-day, recent researches permit us to say that cilia exist without doubt in all the true bacteria, — Bacillus, Bacterium, Spi- rillum. They have been perceived in a great number of forms, — Spirillum volutans, Sp. undula, Vibrio rugula, Spiromonas Cohnii, Vibrio ser- pens, and several species of Bacillus. It is only in the smallest of the bacteria that it has hitherto been impossible to demonstrate their presence. They have, however, been recently seen by Dai- linger and Drysdale in Bacterium termo. Warm- ing has perceived as many as two or three on one extremity in Ophidomonas sanguined Spirillum vohitans var. robustum, and Vibrio rugula. PLATE I. Taken ft om "Monthly Microscopical Journal," of Sept. 1st, 1875. FIG 1. — a. B. termo magnified with the same power as &, which is a specimen of Spirillum volutans, showing flagella at each end. FIG. 2. — B. termo, as seen with a power of about 600 diameters. FIG. 3. — The same as seen with -fa and second eye-piece (3,700 diameters) . FIG. 4. — B. termo, seen with flagellum at one end, the light com- ing in the direction of the arrow. FIG. 5. — The same object when it moved at right angles to its former position, the light coming from the same direction, causing the sight of the flagellum to be lost. FIG. 6 represents one B. termo which was in a still condition, but one flagellum moving. The light came 'in the direction of the arrow. When the end marked 2 b was in focus, a flagellum was seen, but none at the end c. When the end marked l.o was fo- cused carefully, the flagellum at that end was seen, and lost at the endd. FIG. 7. — The true form of B. termo. FIG. 8. — The form as shown by the " supplementary stage " il- lumination before flagella were found, showing the pointed ter- mination of the body at a,' b. TKe Monthly Microscopical Journal Sept. 1.1875. Plate X f30O cUm.irvi9heei by A.Melsel,lith.. F1AQELLA ON BACTERIUM TERMO- By W. H.Dalhnger. F.R.M.S and J.J. Drysdale ORGANIZATION OF THE BACTERIA. 41 EXTRACT FROM PAPER "ON THE EXISTENCE OF FLAGELLA IN BACTERIUM TERMO," BY W. H. DALLINGER, F.R.M.S., AND J. J. DRYSDALE, M.D., F.R.M.S. " In the summer of 1872, some very fine specimens of £. vohitans came under our notice, and were carefully examined. We were enabled fully to confirm Conn's discovery, and demonstrated repeatedly the presence of a pair of swiftly lashing flagella. The drawing at b, Fig. I, was made from a specimen magnified 1,300 diameters (diminished by £). " Having closed for the present our Monad researches, we have been stimulated by the hope that the experience gained by these might enable us to prosecute similar investigations into the true life history of bac- teria. We have commenced the work this summer, and, guided by the analogy of S. volutans, we have been led to make several continuous efforts to find whether or not there existed a flagellum or flagella in B. termo. The task, of course, under the best circumstances, must be a difficult one, from the extreme minuteness of the object. We tried each of Powell and Lealand's powers successively, from the i\ to the -fa, but with no definite result. Repeatedly we both saw vortical action at both the distal and proximal end of the termo, but could not absolutely see the organ causing it. But in the process of our investigations we made very close and careful observations on the fission of this form : we do not purpose now to describe the process, but merely to point out a phenomenon that further confirmed our suspicion of the presence of an invisible filament. In separating into two, the jointed rod of sarcode which is in process of division shakes to and fro at the constriction, as if the constricted part were a hinge ; and at length a clear separation takes place to quite the length of the original termo (sometimes longer), and there is no visible connection between them ; nevertheless they act as one crea- ture, so that if one moves in any direction, the other goes with it, just as the two parts did before separation; showing that, although we cannot see the con- nection, there must be one ; and the presumption was that it was a fine filament, such as we detected in the fission of some monads.1 We could make no further progress in the question apparently ; but our attention was called to the new |th objective prepared by Messrs. Powell and Lealand, with which we were soon supplied. We used it at first with the 'supplementary stage' for very oblique illumination, supplied by the same makers, and this has the advantage of throwing the light in only from one direction. We were soon convinced of the exquisite per- formance of the glass when used as an immersion. Amphipleura pellucida was not merely seen to be striated clearly and sharply, but the striae 1 " M. M. J., vol. x., p. 55 ; and vol. xi., p. 8." 42 MORPHOLOGY OF THE BACTERIA. were resolved into beads with the third and fourth eye-pieces. In like manner the fine striae in Surirella gemma were instantly shown to be beaded, with perfect and brilliant definition, with the second eye-piece. Navicula rhomboides and an extremely delicate specimen of Pleurosigma aifrnuatum which had resisted everything below a ^th immersion, showed beaded striae perfectly. We were therefore encouraged to try again to discover flagella in the termo. Some of our specimens, nourished in Cohn's nutritive fluid, were placed in a drop of distilled water, and put upon the supplementary stage on an ordinary slide covered with the thinnest cover. The utmost delicacy and tact in manipulation of the light is the great desideratum ; but, with this, enough may be secured to work with the fourth eye-piece. The light may be made to enter the objective at almost every angle, but it is always projected in a direction at right angles to the stage ; and the first thing we observed when the objects were sufficiently slow in their movements, and at right angles to the light, was that the ends of the termo, which we (and all other observers, as far as we know) had taken for round, proved themselves to be conical, terminating in a sharp point. The usual appearance of B. termo, as seen with a magnification of about 600 diameters, is seen in Fig. 2 ; whilst the same seen with a magnifying power of 3,700 diameters (^th and second eye-piece) is seen in Fig. 3, where a globular granule is seen in the end of each half. But with the method above referred to, the best condi- tions being secured, the two ends of the bacterium were distinctly pointed, en at a b, Fig. 8, and after nearly five hours of incessant endeavor a flagellum was distinctly seen at one end of each of two termos which moving slowly across the field. The discovery was not sudden and transient, but lasted for at least twenty minutes. The exquisitely delicate flagellum was lashing rapidly the whole time ; and one of its frequent conditions is shown in Fig. 4, the arrow indicating the direction of the light : but if the termo turned round at right angles, as in Fig. 5, all trace of the flagellum was gone, showing that its discovery depended entirely, all things being equal, upon its position in regard to the light. " But this observation was made only by one of us, the other not being present ; and in pursuance of our plan we determined to see it again, convincing ourselves separately, and then together. After many hours of labor, this was accomplished ; and Fig. 6 shows one of two instances which we both saw together at the same time and in the same instru- ment. It was lying still, obliquely across the field, the light coming in the direction of the arrow. Both ends were not perfectly in focus at the same time, but in focusing the end marked 2 b (Fig. 0) the flagellum Distinctly seen, and was seen also to coil and lash; but no flagellum was then seen at the end c of the same object ; but by bringing it into delicate focus it presented the aspect seen at 1 a (Fig. G), which really represents the same object at the same time, only with the other end in the focus, while the end marked d corresponding to 2 b of Fig. 6 was in its turn slightly out of focus, and the flagellum lost to view. This ob- servation, made together, was as satisfactory as could be desired ; and it ORGANIZATION OF THE BACTERIA. 43 was thus demonstrated that there was a flagellum at both ends of the or- dinary B. termo. " It will of course be understood that it is by no means an easy matter to secure the demonstration ; and whenever we repeat it, it must always be with nearly the same amount of trouble and patience, although we can now with the ordinary condenser detect the vortical action, both in front and (occasionally) behind the termo, as we never did before. But the demonstration of the ultimate structure of a fixed object — as for instance Amphipleura pellucida — must be looked upon as a matter of great ease in comparison ; and that for many reasons, the foremost being the motion and the minuteness of the object, the swift play of the flagella, their similarity in optical properties to the fluid in which bacte- ria live, the difficulty of retaining them in focus, and of getting them in such a position in relation to the light as to make demonstration possible. Of course, all this would be removed if deatt bacteria would answer, but they very rarely (indeed only once) have done so with us. The flagel- lum needs to be in slow motion to properly show itself ; for even with the more delicate and minute monads it is a difficult thing to show the flagella in dead forms, since in the majority of cases they appear to be attracted round the body of the creature." § 2. — OF THE DIFFERENT MODES OF GROUPING OF THE BACTERIA. The bacteria are found in different media in two states, — free, isolated (unicellular bacteria), or united several together, either in chains, in filaments, or in masses of greater or less extent, and sometimes by the aid of a mucous substance in which they are imbedded. The free unicellular bacteria are found in the Spirillum, Bacillus, Monas, etc. When they are united, they are grouped in the following modes : — I. Form of a little chain: Torula, Leptothrix. — The usual method of multiplication among the bacteria is by fission (" scissiparite") ; each cor- puscle divides transversely, and gives birth to two 44 MORPHOLOGY OF THE BACTERIA. new individuals, which sometimes become sepa- rated completely the one from the other, to form unicellular bacteria, sometimes remain united; and segmentation again occurring in each portion, a chain is formed of articles more or less numerous. When these chains are formed of spherical bac- teria, they have been called torulce; if they are formed of filiform bacteria, they correspond to leptothrix (Robin). The morphological difference between the torula and the leptothrix consists in the fact that in the first the articles are separated by constrictions, while this is not the case in the second. It is also to be remarked, according to Cohn, that the microbacteria never take either of these forms. Warming states, however, that he has met the torula form in Bacterium lineola, B. catenula, and B. termo (?). Billroth has called these two forms of bacteria streptococcos and streptobacteria. He has even considered it necessary to create the words diplo- coccos and diplobacteria for organisms constituted only of two articles. 2. Form of Zooglcea. — Generally, when bacte- ria are rapidly multiplying, they remain grouped in masses, swarms, or Zooglcea. In the latter con- dition, they are closely pressed against each other in the midst of a viscous substance, hyaline, ho- mogeneous, colorless, and constituting masses more or less diffused or definite, in irregular globes, bunches, or tubes, swimming in the water or near its surface. When the bacteria multiply abundantly, the cells become removed from each ORGANIZATION OF THE BACTERIA. 45 other, so as to leave between them greater inter- vals. The masses sometimes attain a diameter of several centimetres. The gelatinous substance in which the bacteria are included seems to be produced by a thicken- ing and jellification of this cell-membrane, or by a secretion from their protoplasm, but the latter view seems more plausible than the former (De Lanessan). It is commonly the spherical bacteria (Micro- coccus) and the microbacteria (Bacterium) which are found in this state. The filiform bacteria and the spirilla are never found in gelatinous masses (Cohn). Ray-Lankes- ter, however, claims to have met the Spirillum tenue, in the form of zoogloea, and Klein the Spi- rillum undula and rosaceum (Warming). The form of Zooglcea, properly so called, gelat- inous and thick, has never been found by Warm- ing in infusions of sea-water. According to the terminology of Billroth the zooglcea are called gliacoccos and gliabacteria (from yXi'a, mucus substance). 3. Form of My coder ma. — In certain cases, the bacteria unite on the surface of the water, or of the liquid in which they are developed, to form a thick layer, a sort of membrane. This production called my co derma by Pasteur is a sort of zooglcea, but differs from it by the absence of the interme- diary mucous substance. The bacteria are, how- ever, motionless, although living, since they come to the surface to be in contact with oxygen, which is necessary to them. 46 MOEPHOLOGY OF THE BACTERIA. The petalococcos and petalobacteria of Billroth correspond with the mycoderma of Pasteur. 4. Swarms. — We have seen that the filiform and spiral bacteria do not, usually, form zooglwa. These microphytes are either disseminated and free, or united in swarms. This formation may be seen, for that matter, in all the bacteria, when, thanks to abundant nourishment, they multiply rapidly and gather together in considerable masses. They are very active in these sw*arms, whilst in the zoogloea the corpuscles are motionless, because of the intermediary glairy substance. Pulverulent precipitate. — When the nutritive elements are exhausted in a liquid, the bacteria cease to multiply, fall to the bottom of the recep- tacle, and the liquid gradually becomes clear. The deposit formed in this manner may acquire a thick- ness very appreciable to the naked eye. The bac- teria which form this precipitate are not dead, but in a state of temporary repose ; and if a new sup- ply of nutritive material is added to the liquid, they are seen to multiply anew, until this has been exhausted (Cohn). PLATE II. ••• > « - i \ 2 &ti , ••>•.:. ^:\ •"•:." P ' i "•• ' | . '<• '• ^ ' v- ' • • : - '*• ';\v-.-u,.- /. • -. ' ^/W^v,V;. FIG. i. FIG. 2. FIG. 3, FIG. 4. PLATE II. DIFFERENT MODES OF GROUPING. From photo-micographs made in Havana and New Orleans ; copied by permission of the National Board of Health. FIG. 1. — Torula form of spherical bacteria (Micoderma aceti Pasteur) from rotten banana, New Orleans, April, 1880. X 1000 diameters by Zeiss's ^ in. objective. FIG. 2. — Zooglozaform of spherical bacteria developed in culture- cell containing blood of leper. X COO diameters. FIG. 3. — Mycoderma, from surface of foul gutter-water. New Orleans, April, 1880. X 400 diameters by Beck's \ in. objective. FIG. 4. — Leptothrix form of desmobacteria developed in yellow- fever urine, exposed in laboratory, Havana, July, 1879. X 400 diameters by Beck's £ in. objective. CHAPTEE II. CLASSIFICATION OF THE BACTERIA. § 1. — POSITION OF THE BACTERIA. THE place of the bacteria in the scale of beings, for a long time undetermined, demands to be established with precision ; not only for the natu- ralists, who only view the question from a system- atic point of view, but above all for the biologists who study the role of these organisms in the chem- ical or pathological phenomena with which they are associated. According to Ch. Robin, not to define the animal or vegetable nature of these organisms, " is for them as grave as it would be for a chemist to leave undecided the question as to whether it was nitrogen or hydrogen, urea or stearine, which he had obtained from a tissue, or of which he is following the combinations in certain operations." This determination is, to-day, possible ; and, if there are still some differences of opinion among naturalists as to the place of the bacteria among the cryptogams, there is but one opinion as to their vegetable nature. It is surprising to see a savant like M. Pasteur " not to pronounce positively upon the vegetable CLASSIFICATION OF THE BACTERIA. 49 or animal nature of several of the ferments which he has studied/' and of which some belong to the bacteria. We shall first indicate rapidly the characters which permit us, at first, to recognize certain spe- cies of bacteria as organized beings, to determine if they are animal or vegetable, and finally to classify them either among the algae or among the fungi. Distinction of Bacteria from Inorganic Sub- stances. — The question as to whether bacteria are organized beings can only be raised in relation to the smallest species, those Micrococci which are scarcely perceptible with the highest powers ; the organized nature of the other organisms of the same group has never been questioned, even by the earliest observers, who all, since Leeunhoeck, have, without exception, taken them for animals or vegetables. But the smallest forms of bacteria may be confounded with various matters, with organic particles, molecular granules, fa.t globules, etc. " These productions, which are found in con- siderable quantity in the liquids or in the tissues of animal or vegetable origin, often resemble so closely, in form, size, and grouping, the spherical bacteria, that it is absolutely impossible to guard one's self against confusion, unless the most . mi- nute precautions are taken in making the observa- tions " (Cohn). The detritus, the amorphous powder of precipi- tated molecules of inorganic substances, even when 50 MORPHOLOGY OF THE BACTERIA. they exhibit the brownien movement, are easily enough distinguished from Micrococci by optical signs, tiieir angular form, their less refractive power, and finally by their reaction with certain chemical agents; above all if they are mineral substances, crystalline bodies, etc. It will not be the same with molecular granules of organic nature. They have as common charac- ters, their rounded form, their notable refractive power, movements. Nevertheless, their form is less regular, more angular, their color variable, their refractive power always less. In doubtful cases, Tiegel has given a method which enables us to dis- tinguish them from Micrococci. It consists in warming the glass slide which supports the cor- puscles under examination ; if they are " Coccos" they are seen to move in a manifest manner. This does not occur in the case of molecular gran- ules. It is these productions which render it very difficult to observe the phenomena which occur during the coagulation of milk. The caseine sep- arates in the form of extremely minute globules having a very rapid molecular movement. But we may distinguish these from bacteria by the use of liquor potassae, which dissolves the former without attacking the latter. As another example of pseudobacteria, I will mention, after Cohn, the form which fibrine as- sumes when it separates from the plasma of the blood. It disposes itself in very slender filaments, closely resembling filamentous bacteria. CLASSIFICATION OF THE BACTERIA. 51 Fat globules, which are found of all sizes, are, often of the same dimensions as Micrococcus, and are very difficult to distinguish from the latter. The difference in refractive power is slight, and the action of re-agents, such as ether, is not cer- tain in mucilaginous solutions. Hiller, who has paid especial attention to the means of recognizing bacteria, divides them into two groups : — A. The optical signs : comprising 1. The charac- teristic vegetable form, rods, leptothrix, this he recognizes as of little use, as in this case there is no doubt; 2. The characteristic movements of the monads ; 3. The mode of growth and of multipli- cation ; 4. The mode of junction of the granules. B. The chemical signs : 1. False zooglcea become softened and diffluent under the action of liq. potassse, and are coagulated by the direct applica- tion of alcohol ; 2. In sections of tissues, after an hour of maceration in liq. potassse, diluted TVtih> the monads are colored brown by iodine, while fat granules are not. But, in truth, the method of cultivation, ex- tolled by Cohn and Wolff, is .the best means of distinguishing the bacteria. " The distinction of pseudobacteria," says the first of these authors, " from veritable globular bacteria is a problem which our microscopists cannot resolve, in every case, with the desirable certainty. It is only by a study of their mode of development that this distinction can be made. The globules which di- vide and develop in form of chains are organized beings ; when this does not occur, we are dealing with pseudobacteria." 52 MORPHOLOGY OF THE BACTERIA. This is not, however, exactly the opinion of Nageli, who seems to consider movement as the surest distinctive characteristic. "There are," he says, "but three distinctive signs which enable us to recognize with some certainty that granules under observation are or- ganisms, — spontaneous movement, multiplication, and equality of dimensions, united with regularity of form. " The most certain character is movement in a straight or curved line, — a movement which inorganic granules never present. One should take care not to be deceived by movements which are caused by currents in the liquid under observation. Nor should one allow himself to be deceived by the tremulous motion, called molecu- lar movement, in which the granules do not really change their position. These movements are seen in most cells, and even in those of the Schizomy- cetes, and inorganic bodies themselves present it. " Multiplication is a character less important than movement. When among granules some are found united in pairs, it may be supposed with probability that division and multiplication are taking place. When rods are bent at an angle, one may predict their division in two parts. " Finally, as to size and form. Granules of dif- ferent size and of a more or less irregular form ought not to be considered as belonging to the group of segmented fungi ; if, on the contrary, the granules offer dimensions perfectly equal, and a spherical or oval form, the distinction is more CLASSIFICATION OF THE BACTERIA. 53 uncertain : they may belong to the schizomycetes or be of inorganic nature." Place of the bacteria among organized beings. Distinction between animals and vegetables. — The characters which serve to distinguish the inferior animal organisms from the inferior vegetable or- ganisms are of two orders, optical and chemical. A. The optical characters are drawn from the general form, the movements, and the mode of reproduction. The morphological characters have no value except among the larger species of bacteria. If we bring together a Spirillum and a- Spirulina, Kiitz., their affinities will be apparent to every one. It is not the same for the large species of Bacillus, of which, the relations with the Oscilla- toria are evident. The rod form seems very spe- cial, but it does not necessarily imply the vege- table nature of the organisms which possess it. Finally, the spherical bacteria, — Monas and Mi- crococcus, — resemble entirely by their form some infusorial animals. Movement is not a more special character. It is now well proved that it does not belong exclu- sively to animals, and that it is met with in a cer- tain number of the inferior vegetables. In fact, the anatomical characters are not al- ways absolutely reliable ; but it is from these alone that Cohn first, then Davaine, have recog- nized the bacteria as vegetables. B. Chemical characters. Robin depends upon 54 MORPHOLOGY OF THE BACTERIA. these characters to demonstrate the vegetable na- ture of the bacteria. He takes for point of de- parture the notions of general physiology as given by De Blainville in the following points : — 1. We find in animals various elementary sub- stances of the same kind as in plants, and re- ciprocally. 2. The ternary compounds predominate, how- ever, in plants ; and the quarternary, nitrogenized, are more abundant, on the contrary, in animals. 3. In both, the fundamental cellular structure is the same; at least originally for the greater number, and always in the most simple of organ- ized beings, etc. . . . " It results from this, then/' continues M. Eobin, " that so long as there is no digestive tube one can only distinguish plants from animals by the study of their elementary principles, and of the chemical reactions which these exhibit in general ; by the study, in particular, of the reactions which the predominance of ternary cejlulose principles over all others gives to plants, and that of nitro- genized principles in animals, at all periods of their existence." Starting from this basis, Kobin made numerous attempts to find in liquor ammonia, concentrated, as prepared for use in laboratories, a reagent for corpuscles of a vegetable nature. In effect, am- monia dissolves the eggs, the embryos, of all ani- mals, the bodies of all the inferior infusoria, attacks the spermatozoa, etc., whilst it leaves ab- solutely intact all the varieties of cellulose and CLASSIFICATION OF THE BACTERIA. 55 the anatomical reproductive elements of plants, whether it is used cold or boiling. As to the other chemical characters praised during recent years, we will content ourselves with mentioning concentrated acetic acid, which causes all animal tissues to become pale, whilst it is without action on bacteria (Luckonvsky) ; io- dine, and sulphuric acid (Letzerich), etc. Hematoxyline (Luckonvsky) and fuchsin (Hoff- mann) color the bacteria deeply. One ought, then, no longer to give to the bacteria, as do some recent authors, the names of microscopic ani- malcules, — infusoria, microzoa, etc., and other expressions without precision, or consecrating an error. Let us add that some naturalists of high re- pute, Hackel for example, have created for these minute beings, monera, protoplasts, flagellata, dia- toms, etc., an intermediary kingdom between the animal and vegetable, — the Protista. Place of the Bacteria in the Vegetable Series. — The vegetable nature of the bacteria once estab- lished, it remains now to determine to what class of vegetables they belong. Are they algae, or are they fungi ? This is the question which divides the naturalists. It is true that it is to-day very difficult to find a characteristic of these two classes of vegetables, both having, in a general manner, identical forms, similar reproductive apparatus, etc. ; and, if it is impossible to confound a Basidiomycete with a Floridese, for example, it is not the same when 56 MORPHOLOGY OF THE BACTERIA. one studies the inferior species. The only char- acter which appears general is the presence of chlorophyll in the algae and its absence in the fungi. But, if we adopt this distinctive character, and apply it in all its rigor, we are obliged to separate in the inferior algae some forms very nearly related, and which do not differ from their relations except in this particular. And this is ex- actly what happens in the case of the bacteria. In truth, the bacteria, although entirely with- out chlorophyll, have numerous affinities as to form, movement, etc., with the oscillatoriacece, and, according as one or the other of these char- acters have appeared to predominate, the bacteria have been classed as algae or as fungi. It is thus that Davaine, Rabenhorst, then Cohn, struck above all by the resemblance of form, mode of grouping, and of multiplication, have placed the bacteria among the algae. Cohn insists, above all, upon the affinities of the filiform bacteria with the beggiatoa and the leptothrix ; of the micrococ- cus, and of the bacterium, with the chroococcacece. He at first placed them at the commencement of this last series ; but we shall see further on that in his last publications he has disseminated them among the oscillatoriaceae and the chroococcaceae. Robin and Nageli, on the other hand, insist rather upon the affinities of the bacteria with the yeast plants, which are incontestably fungi, and they include them in this class. Robin says expressly : " All the corpuscles de- scribed under the name of Bacterium termo, B. CLASSIFICATION OF THE BACTERIA. 57 punctum, etc.. Zooglcea, Micrococcus, and many others, are vegetable cells, spores of fungi, of sev- eral distinct species certainly; spores, or repro- ductive bodies of the first order, derived one from another, either by germination, fission, or from a mycelium ; reproductive bodies, in a word, of the order of those which Tulasne has arranged under the name of conidia, etc." Nageli establishes in the inferior fungi which produce decompositions three very natural groups. 1. The Mucorini, or mould fungi ; 2. The Saccharomycetes, or budding fungi, which produce the fermentation of wine, beer, etc. ; 3. The ScJiizomycetes, or fission fungi, which pro- duce putrefactive processes. This last group is formed of our bacteria (Micrococcus, Bacterium), etc. Sachs solves the question by uniting the algse and fungi in a single group, the thallophytes, in which he establishes two series exactly parallel, — one comprising the forms with chlorophyll ; the other, the forms which are deprived of it, and preserving in a transverse direction the morpho- logical affinities of these organisms. As this classification is yet but little known, we think it best to give it in the following table : — THALLOPHYTES. Forms with chlorophyll. Forms without chlorophyll. CL. 1. PKOTOPHYTES. A. Cyanophyceae (Oscil- A'. Schizomycetes (Bac- latoriacese, etc.). teria). B. Palmellacese. B'. Saccharomycetes (Ferments). PLATE III. SaccJiaromycetes and Schizomycetes (Nageli), developed in urine (of yellow-fever patient) exposed in laboratory of the Yellow-fever Commission, Havana, July, 1879. Reproduced by permission of the National Board of Health. FIG. 1. — Photo-micrograph made with Beck's £-in. objective and Tolles's amplifier. 400 diameters. FIG. 2. — Photo-micrograph made with Zeiss's ^-in. horn, im. objective. 1,450 diameters. PLATE III. $<*J&J> £§?# ; . 1AM Fi<;. 2. Kcliotyjic I' CLASSIFICATION OF THE BACTERIA. 59 CL. 2. ZYGOSPORE^E. A. Volvocinese. A'. Myxomycetes. B. ConjuguesB and Dia- B'. Zygomycetes. toms. CL. 3. OOSPOKELE. A. Sphseroplese. B. Cceloplastese. Saprolegnise. C. GEdogonise. Peronosporeee. CL. 4. CAEPOSPORE^B. A. Coleochseteae. A'. Ascomycetes. - B. Floridese. B\ QEcidiomycetes. C. CharacesD. C'. Basidiomycetes. Our preferences are for this last mode of classi- fication, but obliged, in the description of species, to follow the classification of Cohn, the most com- plete which has been given hitherto, we must abandon it for the present. § 2. — CLASSIFICATION ; GENERIC AND SPECIFIC CHARACTERS. The numerous classifications of the bacteria of which we have given an abstract in the historical part of this work, show how variable have been the ideas of the microscopists as to the nature of these organisms. Before giving the most recent, those among which we will have to choose, it is best to study the characters upon which authors have depended for grouping the bacteria in genera and species, and to estimate the value of these characters. 60 MORPHOLOGY OF THE BACTERIA. 1. Generic and specific characters. — These have been drawn from the dimensions, form, movement and evolution of the bacteria. The size, which, according to Cohn, is the dom- inating distinctive character, is often indetermi- nable, even in employing the highest powers. Besides, for a great number of neighboring forms, the differences of measurement given as distinctive are so slight that they cannot serve in practice. Thus, according to Dujardin, the Bacterium termo has a length of 1.7 /x, and i\\QB.punctum of 1.7 to 0.6/1. Another difficulty presents itself when we examine bacteria formed of several articles. Shall we consider the length of a single article or of the chain, which consists of a number of articles, a number ordinarily variable ? The form of. the bacteria and their union in colonies, also offer differences, which have been utilized ; but do they depend upon differences truly specific, or do they come from foreign influences, from phases of development of the same organism? Even when one uses these as distinctive specific characters, the form is sometimes of little assist- ance ; since if one refers to the descriptions of Dujardin, the Bacterium termo will be found to have a cylindrical body swollen in the middle, and the B. punctum an elongated ovoid body. As to movement, we have seen that the phenom- ena of mobility or of immobility sometimes pre- sent themselves in the same species, according to age or changes in the medium. We have left, the mode of development, the CLASSIFICATION OF THE BACTERIA. 61 phenomena of reproduction by fission or by spores, as the only character which can serve to establish our natural genera; but, unfortunately, this has only been ascertained for a small number of bacteria, the Bacillus anthracis, for example. The genera of bacteria cannot have the same significance as among animals and superior vege- tables ; they can only be established in accordance with the most prominent characters, reserving the feeble modifications of these generic forms as specific characters. Are there distinct, well-defined, species among the Bacteria ? The microscopists have given the most diverse opinions upon this subject. Miiller, Ehrenberg, Dujardin, Davaine, have admitted the specific dis- tinction of the numerous vibrioniens which they have described. Davaine, however, raises some doubts as to the absolute value of the species established in his time. " Those which are de- scribed to-day by the classifiers," he says, " ought to be considered as the expression of types under which are hidden a certain number of distinct species." Cohn dwells still more upon the impossibility, in which we are to-day, of distinguishing with certainty genera and species among the bacteria. However, he is convinced that the bacteria are di- vided into species as distinctly as the other plants and inferior organisms. It is only the imperfection of our means of observation which makes it impos- sible to recognize these differences. This is above 62 MORPHOLOGY OF THE BACTERIA. all true, he says, of the spirilla, which are not only distinguished from the rod bacteria, properly so called; but which present in their species some differences as constant as any well-defined species of alga or of infusoria. Hallier, Hoffmann, Billroth, Robin, Nageli, etc., consider the different forms of bacteria in a very different fashion. According to them they are not autonomous species, but phases of development of one or of several species. According to Hallier, we may see, a propos of the polymorphism of the bacteria, the singular transformations which he has obtained by their cultivation. According to Billroth, the bacteria belong to a single species of plants, the Coccobacteria septica, with the exception of the Spirillum and Spirochceta, in regard to which Billroth is not willing to give an opinion. This view has been adopted by a certain number of microscopists, and above all by the pathologists, such as Frisch, Tiegel, etc. Robin also admits the genetic relation of Micro- coccus, Vibrio, Bacterium and Leptothrix, but con- siders them the distinct and successive phases in the evolution of several species : 1st. Corpuscles described under the name of Bacterium termo, punctum, etc., Micrococcus ; 2d. Mycelial fila- ments, Vibrio, etc. ; 3. Bacteria, Bacteridies, Micro- bacteria, etc. ; 4th. Leptothrix and forms more advanced. The opinion of Nageli corresponds very nearly with the preceding. " As much as I am con- CLASSIFICATION OF THE BACTERIA. 63 vinced," he says, " that the schizomycetes cannot be grouped in accordance with their action as fer- ments and their exterior forms, and that altogether too many species have been distinguished ; so, on the other hand, it seems to me very improbable that all the schizomycetes constitute a single natu- ral species. " I am rather inclined to suppose that there exists among them a small number of species, which have little in common with the genera and species admitted to-day, and of which each runs through a cycle of determined forms sufficiently numerous. Each of the veritable species of schizomycetes is not limited to presenting itself under the different forms of Micrococcus, Bacterium, Vibrio, and Spi- rillum, but can also show itself as the agent of acidification of milk, of putrefaction, and as the agent producing several maladies." However, Nageli recognizes that it is necessary to distin- guish these forms, notably those of Micrococcus, Vibrio, Bacterium, and Spirillum, without, how- ever, losing from view the fact that the organisms thus classified have a very inconstant constitution, and pass continually from one form to another. Finally, other savants such as M. Pasteur, take less account of the structural characters than of the physiological functions, regarding as a partic- ular species every form of bacterium which is born constantly in a determined medium, or which causes a special kind of fermentation. Nageli opposes to this view the following objections. First, he has verified the presence, in 64 MORPHOLOGY OF THE BACTERIA. the same decomposition, of several different forms of schizomycetes. On the other hand, in decom- positions quite different, we may observe schizo- mycetes entirely similar as to their exterior form. Finally, we may change the mode of action of a schizomycete in subjecting it to a certain treat- ment. One sees that it is truly difficult to form an opinion as to the value of these species purely physiological To sum up, the characters which may be used in order to establish genera and species in the group of the bacteria are of small number and of very unequal value. Some, characters of form, of dimension, of movement, etc., are often difficult to determine, or have only a relative value ; others, characters drawn from development and reproduc- tion, are only known in so few species that they cannot be made to serve as a basis of classifica- tion. One will not be surprised, then, to find that there is no natural classification of the bacteria, and that it is impossible for the naturalists to give one. All those that can be established are pro- visory, being only based upon the morphology of these organisms. Following the example of all the botanists, we will use an analogous classification, but without wishing to prejudge in any particular the genealogical relationship of the different or- ganisms, which we shall consider as distinct gen- era and species. CLASSIFICATION OF THE BACTERIA. 65 § 3. — CLASSIFICATION AND DESCRIPTION OF THE GENERA AND SPECIES OF THE BACTERIA. « We have seen in the historical portion of this work, a propos of the classifications which have been given of the bacteria, that, in 1872, M. Cohn, recognizing the numerous relations, absence of chlorophyll, mode of nutrition, etc., which make these organisms a natural family, divided them into four tribes : — 1. The Spherobacteria, or spherical bacteria. 2. The Microbacteria^ or B. in short rods. 3. The Desmobacteria, or B. in straight filaments. 4. The Spirobacteria, or B. in spiral filaments. In the spherobacteria, Cohn has only adopted one genus, the g. Micrococcus, of which the spe- cies are divided into three series, — the pigmen- tary M., or chromogenes, the M. of fermentations, or zymogenes, and the M. of contagious affections, or pathogenes. The microbacteria include only the genus Bac- terium, with two species, B. termo, Dujardin, and B. lineola, Cohn. The desmobacteria comprehend the g. Bacillus and Vibrio ; the first established by Cohn for the rectilinear filaments is composed of the B. subtilis, Cohn (with B. anthracis as a variety) and the B. ulna, Cohn ; the second, characterized by undu- lating filaments, is reduced to V. rugula and ser- pens, Auct. 5 66 MOEPHOLOGY OF THE BACTERIA. Finally, the spiral filaments of the spirobacte- ria characterize the gr. Spirillum and SpirocJiceta, which might be united in a single genus compris- ing 8p. plicatile, temie, undula,*and volutans. Since then, Cohn, struck with the affinities which each of the preceding genera presents with several genera of oscillatoriacese and of chroococ- ceae, from which the bacteria only differ by the absence of chlorophyll, has established a class of Schizophytes, which includes all the inferior vege- table organisms, provided or not with chlorophyll, multiplying by fission. We give below the complete table : — 2. Classification of the Schizophytes, Cohn. TRIBE 1. — GLJEOGENES. Cells free or united in glairy families by an intercellular substance. A. Cells free or united by 2 or by 4 : Cells spherical . . CHROOCOCCUS, Nag. Cells cylindrical . SYNECHOCOCCUS, Nag. B. Cells united in glairy families, amorphous in state of repose : a. Cellular membrane, con- founded with the intercel- lular substance : 1. Cells without phyco- chrome, very small : Cells spherical . . MiCROCOCCUS, Hallier, CLASSIFICATION OF THE BACTERIA. 67 Cells cylindrical . BACTERIUM, Duj. 2. Cells with phyco- chrome, larger : Cells spherical . . APHANOCAPSA, Nag. Cells cylindrical . APHANOTHECE, Nag. b. Intercellular substance formed of several mem- branes enclosed one with- in the other : Cells spherical . . GLCEOCAPSA, Kg. Cells cylindrical . GLCEOTHECE, Nag. C. Cells united in glairy fam- ilies of definite form : a. Families of a single layer of cells disposed in plates : 1. Cells in fours form- ing a plane surface . MERISMOPEDIA, Meyen. 2. Cells without regular arrangement, forming a curved surface : Cells spherical, fam- ilies with reticu- lated rupture . . CLATHROCYSTIS, Henfr. Cells cylindrical, cu- neiform , families divided by con- striction . . . CCELOSPHLERIUM, Nag. b Families with several lay- ers of cells, united in spher- ical corpuscles : 1. Number of cells de- termined : Cells spherical, col- orless, arranged in fours . . . SARCINA, Goods. 68 MORPHOLOGY OF THE BACTERIA Cells cylindrical, cu- neiform, with phy- cochrome, with- out regular ar- rangement . . GOMPHOSPH^ERIA, Kg. 2. Number of cells very great and indetermi- nate : Cells colorless, very small .... Ascococcus, Billr. Cells colored by phycochrome and larger . . POLYCYSTIS, Kg. COCCOCHLORIS, Spr. POLYCOCCUS, Kg. TRIBE 2. — NEMATOGENES. Cells disposed in filaments. A. Filaments not branched : a. Filaments free or inter- laced. 1. Filaments cylindrical, colorless, articulations not very distinct : Filaments very slen- der, short . . . BACILLUS, Cohn. Filaments very fine, long LEPTOTHRIX, Kg. Filaments larger, long .... BEGGIATOA, Trev. 2. Filaments cylindrical, with phycochrome, articles well defined, without cellular re- production . . . HYPHEOTHRIX, Kg. OSCILLARIA, BOSC. CLASSIFICATION OF THE BACTERIA. 69 3. Filaments cylindrical, articulated, with co- nidia : Filaments colorless CRENOTHRIX, Cohn. Filaments with phy- cochrome . . . CHAM^BSIPHON. 4. Filaments spiral without phycochrome : Filaments, short, light, sinuous . VIBRIO, Ehr. Filaments, short, spi- ral, rigid . . . SPIRILLUM, Ehr. Filaments, long, spi- ral, flexible . . SPIEOCH^TE, Ehr. with phycochrome : Filaments long, spi- ral, flexible . . SPIRULINA, Link. 5. Filaments in chaplet : Filaments, without phycochrome . . STREPTOCOCCUS, Billr. Filaments with phy- ) ANAB^NA, Bory. cochrome . . . i SPERMOSIRA, Kg. 6. Filament flagelliform, slender MASTIGOTHRIX, etc. b. Filaments united into glai- ry families by an intercel- lular substance : 1. Filaments cylindrical, colorless MYCONOSTOC, Cohn. 2. Filaments cylindri- 1 CHTHONOBLASTUS. cal, with phyco- LlMNOCLIDE, Kg. . chrome . . . . ; 3. Filaments in chaplet . NOSTOC, etc. 4. Filaments flagelliform, slender RIVULARIA, etc. 70 - MORPHOLOGY OF THE BACTERIA. B. Filaments with false ramifi- cation : 1. Filaments cylindri- j CLADOTHRIX, Colin, cal, colorless . . ) STREPTOTHRIX, Cohii. 2. Filaments cylindri- ) „ cal, with phyco- CALOTHBIX, Ag. chrome .... ) SCYTONBMA, Ag. 3. Filament in chaplets . MERIZOMYRIA, Kg. 4. Filaments flagelli- form, slender towards the extremity . . SCHIZOSIPHON, Kg. GEOCYCLUS, Kg. An inspection of this table shows that each of the genera of the ancient group of the bacteria has been placed beside some genus of oscillatori- acese, which it resembles by its organization, — Micrococcus and Bacterium, beside Aphanotkece and Aphanocapsa; Bacillus, beside Leptothrix and Beggiatoa ; Vibrio and Spirillum, beside Spi- rulina. These affinities are undeniable, and the advan- tages of such a classification are manifest ; but, in a work like this, we cannot think of employing it. We preserve, then, in a distinct group the schizo- phytes deprived of chlorophyll, which may be arranged in the four primary divisions of Cohn with the exception of Sarcina, Ascococcus, Creno- thrix, etc., and the other genera created recently by this botanist. Thus we will describe successively : — 1. The Spherobacteria of Cohn ; and beside them the different Monas recently studied, — the Micrococ- CLASSIFICATION OF THE BACTERIA. 71 cus described by Hallier in several infectious mal- adies. 2. The Microbacteria. 3. The Desmobacteria, including Bacillus, Lepto- tJirix, Beggiatoa, and Crenothrix. 4. The Spirobaeteria, including the three genera, Vibrio, Spirillum, and Spirochceta. 5. Finally, we will give some account of the Mer- ismopedia, Sarcina, Ascococcus, Streptococcus, Myco- nostoc, Cladothrix, and Streptothrix. 1. SPHEROBACTERIA, Cohn. The spherical bacteria are characterized by their rounded or oval form, their small size, often less than 1 ju,. They are ordinarily isolated, often in pairs (diplococcus), sometimes in a chain of several articles (streptococcus = torula of Cohn), the my- cothrix of Hallier and Itzigsohn, or in the form of zooglcea when they are young and actively multi- plying, and that of mycoderma, when they are gathered upon the surface of liquids. They have no spontaneous movement, but a simple molecular trepidation. Functions : " The spherical bacteria are fer- ments, not producing putrefaction, but substitu- tions of another kind" (Cohn). 06s. According to the facts observed by Koch, Cohn, Pasteur, Toussaint, upon the development of certain bacteria, it is very probable that some at least of the spherobacteria are spores of Bacil- lus or of other bacteria ; at least, the micrococci and these spores are identical in form and aspect. 72 MORPHOLOGY OF THE BACTERIA. The spherobacteria include only the genus Mi- crococcus. g. Micrococcus, Cohn (Hallier emend. — Micro- sphceria, Cohn, ante). Cells colorless, or scarcely colored, very small, globular or oval, forming by transverse division filaments of two or several articles, in form of chaplet, or united in numerous cellular families, or in gelatinous masses, all deprived of move- ment. The species are divided into three physio- logical groups: — a. M. Chromogenes. b. M. Zymogenes. c. M. Pathogenes. SECTION (A) : MICROCOCCUS CHROMOGENES. The pigmentary bacteria grow in the state of Zooglcea upon the surface of the substances which furnish them with nutriment. They are always alkaline ; all are avid of oxygen ; their morphological characters are identical, and one can only distinguish them by their different coloring properties. According to Cohn, they are veritable spe- cies ; for 1. Their pigments offer the greatest diversity as to chemical action and by spectro- scopic analysis, etc. ; 2. Each species cultivated in the most diverse media produces always the same coloring matter. CLASSIFICATION OF THE BACTERIA. 73 They are divided into two categories, accord- ing as the pigment is soluble or not in water. 1. Coloring matter insoluble. M. Prodigiosus, Cohn (Monas prodigiosa, Ehrb. ; — Palmella prodigiosa, Mont. ; — Bacteridium prodigiosum, Schroeter). A red gelatinous mass, pink carmine, develop- ing upon cooked alimentary substances placed in damp air, never before cooking. It has also been observed in red milky at- tributed incorrectly to lesions of the teats, etc. (Cohn). M. luteus, Cohn (Bacteridium luteum, Schroeter). A yellow gelatinous mass studied by Schroeter and Cohn upon potatoes. 2. Coloring matter, soluble. M. aurantiacus, Cohn (Bacteridium auriantiacum, Schroeter). Little drops, or stains, more or less extended, golden yellow, cultivated by Schroeter, upon slices of cooked potato ; by Cohn, upon hard white of egg. M. chlorimis, Cohn. A glairy yellowish-green pigment found upon hard white of egg, not reddened by acids, but loses its color. M. cyaneus, Cohn (Bacteridium cyaneum, Schroe- ter). Pigment deep blue, observed by Schroeter 74 MORPHOLOGY OF THE BACTERIA. upon cooked potato, and cultivated by Colin in nutritious solutions. This coloring matter is reddened by acids, and restored to blue by al- kalies, just as that which forms when lichens are macerated in presence of ammonia. M. violaceus, Cohn (Bacteridium violaceum, Schroe- ter). Violet-blue masses or glairy stains formed of elliptical corpuscles larger than those of M.pro- digiosus, observed first by Dr. Schneider, then by Schroeter on cooked potato. Later, Cohn has described the two following new species (1876), which should be included in this group : M. Candidas, Cohn. Stains and points ichite as snow, observed upon slices of cooked potato. M. fulvns, Cohn. Little rust-colored drops, consisting of cells, globular or united in pairs, in a tenacious inter- cellular substance, diameter 1.5 ^ observed by Eidam, then by Kirchner, upon horse dung. It is also to the genus Micrococcus that we must refer the little globular bacteria, gifted with movement, found by Eberth in white, yel- low, and red perspiration, and by Chalvet in the pus on the edges of certain wounds, but which should not be confounded with the blue color produced by a Bacterium. CLASSIFICATION OF THE BACTERIA. 75 SECTION (B): MICROCOCCUS ZYMOGENES. Globular bacteria producing fermentations of diverse nature. M. crepnscuhim, Cohn (Monas crepusculum, Ehrb.). Globular cells, colorless, developing in all in- fusions of animal and vegetable matter under- going decomposition. M. ureae, Cohn. Oval cells, isolated, diameter 1.5 ^ (Pasteur), 1.2 to 2 p (Cohn) or united by 2, 4, to 8 (to- rulci), in a line, straight, curved, zigzag, or even in cross form. In urine, of which it transforms the urea into carbonate of ammonia (Pasteur). A Torula which appears identical with the preceding Micrococcus, produces the decomposi- tion of hippuric acid into benzoic acid and gly- collamine (Van Tieghem). M. of stringy wine, etc. Globular cells of 2 /x diameter, in chaplets, found in stringy wine, perhaps identical with the preceding (Pasteur). A Torulacese quite similar is found in certain fermentations of tartrate of ammonia and of beer yeast, with or without the addition of car- bonate of potash (Pasteur). SECTION (c) : MICROCOCCUS PATHOGENES. Spherical bacteria found in affections of a con- tagious nature. 76 MORPHOLOGY OF THE BACTERIA. M. vaccinae, Cohn (Micros^)hcera Vaccince, Cohnj. Very small micrococci, = 0.5 /x scarcely, iso- lated or united in pairs in recent vaccine virus and in the pus of variola pustules. By cultiva- tion, chaplets of from two to eight cells may be obtained, then masses containing sixteen to thirty-two cells of 10 p, and more diameter. The M. of vaccine virus and of variola are identical, and Cohn regards them as different races of the same species. M. diphtheriticus, Cohn. Granular cells, ovoid, measuring from 0.35 to to 1.1 //,, isolated or more often united in twos or in a chaplet of four to six cells ; sometimes multiplying in colonies and extending them- selves in all the diseased tissues, decomposing and destroying them ((Ertel). M. septicus, Cohn (Microsporon septicus, Klebs). Little rounded cells, of 0.5 /*, motionless and crowded in masses or united in chaplets, in the secretion of wounds in cases of septicemia (Klebs), in zooglcea in callous ulcers, in isolated cells, united in pairs, or in chaplets in the se- rum of epidemic puerperal fever (Waldyer), in all the tissues, vessels, etc., in cases of pyemia and septicemia. M. bombycis, Cohn (Mycrozyma bombycis, Be- champ). Cells with a diameter of 1 ^, ordinarily united in chaplets of two, three, four, five, or more, in CLASSIFICATION OF THE BACTERIA. 77 the intestine of silkworms sick with "laflach- erie" (Pebrine). In a more recent work, Cohn (Beitrage, 1875, p. 201) gives them an oval form and a diameter of 0.5 p, at the outside. Hallier has described many other Micrococci in diverse contagious or virulent affections. We will only refer to them in a summary manner : } — M. of the variola of animals, Hallier. Small M. endowed with active movement, furnished with a very delicate caudal append- age, sometimes united in the form of little elon- gated rods, found in pustules, spontaneous or inoculated, in the lymphatic canals and the gan- glia of animals attacked with variola. M. of rugeola, Hallier. Very small colorless M., having often a caudal prolongation, in the sputa and blood of the sick. M. of scarlatina, Hallier. M., free or in colonies, on the surface or in the interior of the blood corpuscles, or in chains. M. of epidemic diarrhoea, Hallier. M. in intestinal matters with vibrios, cells, and monads. (?) 1 "It is quite probable that Hallier comprises, in part, under the name of Micrococcus the same organisms that I call spherical bacteria ; but the doctrine of Hallier concerning Micrococcus, as has already been pointed out by Hoffmann and de Barry, is so covered by inexact asser- tions and improbable hypotheses, that it is impossible to draw any con- clusions from the facts he has observed." — COHN, Beit. II, p. 148. 78 MORPHOLOGY OF THE BACTERIA. M. of exanthematous typhus, Hallier. M. relatively large, brown, having a rapid movement, sometimes in chains (Mycothrix), in the blood. M. of intestinal typhus, Hallier. M. very small, in repose in the blood ; larger, endowed with active motions, and furnished with contractile appendices in the dejections. M. of glanders, Ziirn. Cells free or attached to the blood globules, or even penetrating into their interior, sometimes in chains (Mycothrix) in the blood. M. in chains, very numerous, and endowed with rapid movements, in the lymphatic ganglia, the mu- cus of the frontal sinuses, and in the chancroid ulcers. M. of syphilis, Hallier. M. numerous, colorless, free or in globules, in gonorrhoea, the primitive ulcer, and the blood of persons suffering from constitutional syphilis. MONADS. Beside the Spherobacteria are placed the Mon- ads, not the organisms described under this name by the older microscopists, comprising micro- phytes, spores, and infusorial animals, but the Monas as understood by botanists of the present day. Thus limited, the Monads include also, be- sides some microphytes related to the Spherobac- teria, and differing from them by their greater dimensions, some organisms of doubtful affinities. CLASSIFICATION OF THE BACTERIA. 79 As in the case of the Micrococci it is very probable that the Monads are only the spores, or lower forms of bacteria higher in the scale. Cohn places the Monas vinosa of Ehrenberg with the Clathrocystis roseopersicina, Cohn (Bacterium ru- bescens, Kay-Lank.), considering it a spore of the latter. Monas vinosa, Ehrb. Cells spherical, oval, regular, of 2.5 /JL, often united in pairs, formed of a pink substance with granules of a deeper color, having spontaneous movements. Hob., waters containing decomposing vegetable matters (Ehrb. 1838, Ch. Morren 1841, Perty 1852, Cohn 1875). M. Okenii, Ehrb. Cells cylindrical; average length T to 15 ^ (Cohn), 10 /u, (Ehrb.), sometimes from 60 to 80 //, (Warming), diameter 5 //, ; of a beautiful red color, having a rapid gyratory movement, with a cilium at the posterior extremity or two cilia at the two extremities. Hob., stagnant water (Ehrb. 1836, Eichwald, Weiss, Cohn, etc.). M. Warmingii, Cohn. Cell cylindrical, pink, containing at its two rounded extremities some deep-red granules ; length 15 to 20 //., width 8 fj, ; movement uncertain, having a vi- bratile cilium. Hab.^ brackish water on the coast of Norway (Warming). M. gracilis, Warming. Cells straight, cylindrical, pink, rounded at the two extremities ; length 60 //., thickness 2 //, ; move- ment slow. Hob.) fresh water. 80 MORPHOLOGY OF THE BACTERIA. Ehdbdomonas rosea, Cohn. Cells pale pink, isolated, fusiform ; eight times as long as broad, having a length of 20 to 30 /^, and a width of 3.8 to 5 /JL; having a slow oscillatory move- ment, the pink substance containing numerous gran- ules of darker color and vacuoles. Hob., stagnant water. Ophidomonas sanguined, Ehrb. Cells pale pink, spiral, rigid, movement active ; thickness 3 p, length of one turn of the spiral, 9 to 12 p. Hab., brackenish waters of Denmark (Warm- ing). Spiromonas Cohnii, Warming. Cells spiral, flattened ; 1 \ turn of spiral, diam. 1.2 to 3.5 /*, width 1.2 to 4 p. ffab., coast of Denmark. 2. MlCROBACTERIA, Cohn. Rod-bacteria, cells cylindrical, short, having spon- taneous movement. A single genus, — Bacterium. g. Bacterium, Duj. emend. Cells cylindrical or elliptical, free or united in pairs during their division, rarely in fours, never in chains (leptothrix or torula), sometimes in zooylcea (differing from the Z. of spherical B. by a more abundant and firmer intercellular substance), having spontaneous movements, os- cillatory and very active, especially in media rich in alimentary material and in presence of oxygen. CLASSIFICATION OF THE BACTERIA. 81 We might, as in the Spherobacteria, divide the rod-bacteria into three groups : 1. the bac- teria of putrefaction, B. termo, B. lineola, and their varieties ; 2. the Bacteria of the lactic and acetic fermentations, etc. ; 3. Chromogenes, B. of colored milk and pus. B. termo, Ehrb. 1830, Duj. ( Vibrio lineola, Ehrb. ex. p. 1838; Monas termo, Miiller). Cells cylindrical, slightly swollen in the middle, isolated, sometimes united in pairs, two to five times as long as wide; length 2 to 3 /A, thickness 0.6 to 1.8 fju ; movements oscillatory. Appears at the end of a very short time in all infusions of animal and vegetable substances ; multiplies with rapidity in numerous zooglcea ; then disappears as other species, to which it serves as nutriment, are developed. According to recent observations, this bacterium has cilia (Dallinger, Drysdale, Warming). Warming has also found it in the state of torula. B. termo is the veritable agent, the first cause, of putrefaction, it is the true ferment saprogene (Cohn). M. Warming has recently described two allied forms : — B. griseum, cells larger, more rounded ; length 2.5 to 4 /*, thickness 1.8 to 2.5 /z. In infusions of fresh and salt water. B. littoreum, cells elliptical or elongated, slightly rounded ; length 2 to 6 /LI, thickness 1.2 to 2.4 p. Coasts of Denmark. B. lineola, Cohn ( Vibrio lineola, Ehrb. ex p., Duj., Miiller, V. tremulans, Ehrb., Bacterium triloculare, Ehrb). 82 MORPHOLOGY OF THE BACTERIA. Cells cylindrical, straight, rarely a little twisted, larger than the cells of B. termo, isolated or united in pairs, sometimes in fours, never more ; length 3.8 to 5.25 /*,, thickness attains 1.25 //, ; movements like those of B. termo, but a little more active. Is found in various vegetable and animal in- fusions of fresh or salt water, often takes the form of zoogloea containing motionless rods in their mucus substance. Warming has met it in the form of chains composed of eight to ten cells (torula). Its protoplasm is dotted with re- fractive granules. It is not known whether B. lineola constitutes a specific ferment (Cohn). The B. fusiform, Warming, differs from the preceding by the form of its body, which is attenuated at the two extrem- ities ; length 2 to 5 p., width 0.5 to 0.8 p ; plasma not punc- tated. Beside these species, which have been well studied, may be placed the following, which demand new investigations : — B. punctum, Ehrb. Elongated rods, oval, colorless, having slow movements, oscillating, often united in pairs; length 5.2 /x, thickness 1.7 /*. Diverse infusions of animal substances. B. catenula, Duj. Body filiform, cylindrical, often united in three, four, or five ; length 3 to 4 /A, thickness 0.4 to 0.5 p,. In fetid infusions, in typhoid fe- ver (Coze and Feltz). CLASSIFICATION OF THE BACTERIA. 83 Vibrio lactic, Pasteur. "Articles almost globular, very short, a little swollen at the extremities ; length of an article, 1.6 p, of a series, 50 /A." This vibrio seems to resemble B. catenula or B. termo. It is developed, according to Pas- teur, in sweetened liquids, where it causes the formation of lactic acid and the coagulation of the casein of milk. According to other re- searches, coagulation of casein results from the influence of a soluble ferment (zymase), and not from an organized ferment. Acetic ferment (Mycoderma aceti, Pasteur, Ulmna aceti, Ktg.). 44 Articles short, constricted, two to three times as long as broad; length 1.5 /*, often united in long chains, forming pellicles on the surface of a liquid." This species is also very similar to the pre- ceding. It must not be confounded with the Mycoderma vini, which may develop in the same media, but which is a fungus of the group of Saccharomycetes. The acid fermentation of beer seems to be due to a form of Bacterium resembling B. termo (Cohn), but a little larger than the type. Cohn has found it in beer undergoing acid fermenta- tion, beside oval saccharomyces, elliptical bac- teria, having movement, often united in pairs, rarely in fours, etc. Vibrio tartaric right (Pasteur). Bacteria similar to those of the lactic fermentation, PLATE IV. From " Pasteur's Studies on Fermentation." Macmillan fr Co., London, 1879. " The engraving represents the different diseased ferments, together •with some cells of alcoholic yeast, to show the relative size of these organisms." FIG. 1 represents the ferments of turned beer, as it is called. These are filaments, simple or articulated into chains of different size, and having a diameter of about the thousandth part of a millimetre (about YmW inch). Under a very high power they are seen to be composed of many series of shorter filaments, immovable in their articulations, which are scarcely visible. In No. 2 are given the lactic ferments of wort and beer. These are small, fine, and contracted in their middle. They are generally detached, but sometimes occur in chains of two or three. Their diameter is a little greater than that of No. 1. In No. 3 are given the ferments of putrid wort or beer. These are mobile filaments, whose movements are more or less rapid, according to the temperature. Their diameter varies, but is for the most part greater than that of the filaments of Nos. 1 and 2. They generally appear at the commencement of fermentation, when it is slow, and are almost invari- ably the results of very defective working. In No. 4 are given the ferments of viscous wort, and those of ropy beer, which the French call filante. They form chaplets of nearly spher- ical grains. These ferments rarely occur in wort, still less frequently in beer. No. 5 represents the ferments of pungent, sour beer, which possesses an acetic odor. These ferments occur in the shape of chaplets, and consist of the mycoderma aceti, which bears a close resemblance to lactic ferments (No. 2), especially in the early stages of development. Their physiolog- ical functions are widely different, in spite of this similarity. The ferments given in No. 7 characterize beer of a peculiar acidity, which reminds one more or less of unripe, acid fruit, with an odor nui tjtneris. These ferments occur in the form of grains which resemble little spheri- cal points, placed two together or forming squares. They are generally found with the filaments of No. 1, and are more to be feared than the latter, which cause no very great deterioration in the quality of beer, when alone. When No. 7 is present, by itself or with No. 1, the beer ac- quires a sour taste and smell that render it detestable. We have met with this ferment existing in beer unaccompanied by other ferments, and have been convinced of its fatal effects. No. 6 represents one of the deposits belonging to wort. This must not be confounded with the deposits of diseased ferments. The latter are always visibly organized, whilst the former is shapeless, although it would not always be easy to decide between the two characters, if sev- eral samples of both descriptions were not present. This shapeless de- posit interferes with wort during its cooling. It is generally absent from beer, because it remains in the backs or on the coolers, or it may get entangled in the yeast during fermentation, and disappear with it. Among the shapeless granules of No. 6 may be discerned little spheres of different sizes and perfect regularity. These are balls of resinous and coloring matter that are frequently found in old beer at the bottom of bottles and casks. They resemble organized products, but are nothing of the kind. PLATE IV. Heliotype Printing Co.. Boston. CLASSIFICATION OF THE BACTERIA. 85 with globular articles, short ; diameter 1 /*, united in chains of 50 //,. Decomposes racemic acid, causing the right tartaric acid to disappear, and setting free left tartaric acid. MlCROBACTERIA CHROMOGENES. B. xanfhinum, Schroeter ( Vibrio synxanthus, Ehrb.). " Bodies cylindrical, slightly flexible, formed of cor- puscles rarely exceeding five in number ; length of an article, 0.7 to 1 /*. In tainted cow's milk, to which it gives a yellow color." B. syncyanum, Schroeter ( Vibrio syncyanus, Ehrb.). This Bacterium, which has the same charac- ters as the preceding, has been observed in sour milk, to which it gives a blue color. B. cemginosum, Schroeter. In greenish blue pus. These B. chromogenes resemble entirely the lactic vibrios, B. termo or catenula. According to Robin, colored milk contains colorless vibrios, and the coloration is due to an alga similar to Leptomitus. B. brunneum, Schroeter. Rods in a brown coloring matter in infusions of rotten corn. Following the colored Microbacteria, I place two species of Bacterium recently described by Ray-Lankester and Warming. B. rubescenS) Ray-Lank., 1873. Under this name Ray-Lankester has described 86 MORPHOLOGY OF THE BACTERIA. some phases of development of Clathrocystis roseo- persicina of Cohn. Now Cohn is inclined to regard the Monas vinosa, Ehrb. as the wandering cells of Clathrocystis. On the other hand Warming has de- scribed his : — B. sulfuratum, Warming, 1876, giving it for synonymes, Monas vinosa, Ehrb.; M. erubescens, Ehrb.; M. Warm- tn0ru,Cohnj Rhabdomonas rosea, Cohn. It follows, then, that the Monas which we have described with the Spherobacteria should be referred to a Bacterium called sulphuratum by Warming, but which is also identical with B. rubescens of Ray-Lankester. 3. DESMOBACTERIA. Filiform bacteria, composed of elongated cylin- drical articles, isolated, or in chains more or less extended, resulting from transverse division. Un- der this form they correspond to leptothrix, Auct. (differing from torula in that the filaments are not constricted at the point of junction of the articu- lations) ; filaments sometimes united in swarms, never in zoogloea. Movements and state of re- pose alternating and depending upon the presence or absence of oxygen, the reaction of the medium, and other conditions unknown. Some forms never exhibit movement. — Bacteridie of Davaine (Cohn). We will only preserve in the Desmobacteria the genus Bacillus, Cohn. The vibrios are rather al- lied to Spirillum because of their undulating fila- ments. However, after the exposition of the different species of Bacillus, we will say something of three genera of colorless oscillatoriacese, which are nearly CLASSIFICATION OF THE BACTERIA. 87 related to them, — the teptothrix, Beggiatoa, and Crenothrix. 1. Fil. with indistinct articulations Fil. very slender, shorf . . . . BACILLUS. Fil. very slender, long .... LEPTOTHRIX. Fil. thick, broad BEGGIATOA. 2. Fil. articulated distinctly .... CRENOTHRIX. g. Bacillus, Cohn. The bacilli are characterized by slender fila- ments, straight, short or of moderate length, rigid or flexible, endowed or not with motion. One species is chromogene, the B. ruber of Cohn. Finally it is to this genus that we should refer the Amylobacter of Trecul. B. subtilis, Cohn (Vibrio- sub tilis, Ehrb.; Ferment butyrique, Pasteur). Filaments very slender and elongated, formed of a single cell having usually a length of 6 /*, or of two articles of which the total length is then 12 JJL, or of three (length 16 /i), or of a greater number (some- times as many as twenty, with a total length of 40 to 60 and 130 yu,) ; thickness not measurable, well-defined movements of flexion, active or passive, and of trans- lation forward or backward ; reproduction by fission and by globular or oval spores developing themselves in the interior of the articles (Cohn). Is found in stagnant water. Plays a great role in the butyric fermentation (Pasteur). This B. exists in rennet, can support a temperature of 105°, and live in a medium deprived of free oxygen, in which case it takes a form cephalee, containing persistent spores, 88 MORPHOLOGY OF THE BACTERIA. which, when set free, give birth to other rods of Bacillus (Cohn). B. anthracis, Cohn (Bacteridie charbonneuse, Da- vaine). Species very similiar to the preceding, generally longer and always motionless ; length 4 to 12 and even 50 JJL, thickness, scarcely appreciable, 0.8 to 14 //, (Bellinger). The B. anthracis is developed in charbon (malignant pustule of man, sang de rate of sheep, maladie de sang of cattle, fitvre charbon- neuse of horses), and in the rabbit, the rat, etc. ; never in the dog, the cat, the birds, and cold- blooded animals. It is found above all in the capillary vessels. Cultivated in suitable media, such as the aqueous "humor of the eye of the ox, the Bacillus of anthrax develops spores in the interior of its filaments, which may germin- ate and reproduce rods (Koch). According to recent observations not yet published, by cultivating the B. Anthracis in the blood of the dog, a development of veritable sporangia may be obtained, containing from three to six spores (Toussaint). B. amylobacter, Van Tieghem (Amylobacter, Uro- cephalum and Clostridium Trecul). B. occurring, like the preceding, under various forms, — in pointed cylindrical filaments of 6.6 to 26 p in length and 1.1 ^ in thickness, or in form of tadpole, with a spore in the terminal swelling, or of a spindle, with a spore in the middle. In fact, it does not differ from B. subtilis, except by the appear- CLASSIFICATION OF THE BACTERIA. 89 ance of starch in its protoplasm at the end of the period of multiplication. These B. are sometimes endowed with movement (Nylander). It develops in vegetable tissues, which fall into putrefaction, spontaneously according to Trecul, or introduced from without by a mech- anism still unknown. This is the essential agent of vegetable putrefaction (Van Tieghem). B. ulna, Cohn ( Vibrio bacillus, Ehrb.). Filaments articulated, thick, and rigid, formed of one, two to four articles, straight or broken in zig- zag ; length of an article 10 /^, length of a filament of four articles 42 /j, ; slow movements of rotation and of progression. Develops in various infusions of fresh or salt water. In certain cultivations, Cohn has seen large globules (spores ?) form in the protoplasm. Warming believes that he has seen cilia. B. r uber, Cohn. Long rods, isolated or united in two or four, movement very active ; in a red mucous sub- stance, vermillion, developed upon grains of rice. Observed by Franck and Cohn. Davaine has described five additional species of Bacteridies, which appear to be bacilli. They are : — La Bacteridie intestinal. Filaments straight, thick from 10 to 40 fj, in length. In the intestines of birds. La B. du levain. Filaments slender and short, of 10 to 20 /-t, divided 90 MORPHOLOGY OF THE BACTERIA. into two, sometimes three to four articles, identical with the B. of charbon (Davaine). La B. glaireuse. Filaments extremely slender, straight or elbowed ; attaining 10 //, in length. La B. du vin tourne. Filaments very slender, of variable length, flexible, indistinctly articulated. La B. des infusions. Filaments of 10 to 20 /u,. In various infusions. g. Leptothrix, Ktz. The Leptothrix differ from Bacilli by their filaments being very long, adherent, very slen- der, and indistinctly articulated. Their forms are numerous. The following are the principal : — L. rigidula, Ktz. Length 100 to 150 /*, diameter 1.3 to 1.9 /JL. In stagnant water, adherent to other vegetables. L. croespitosa, Ktz. Length 100 to 200 /*, diameter 2.4 to 2.8 /*. Upon humid rocks. L. brevissima, Ktz. Length 75 to 100 /JL, diameter 2.7 to 3.5 p. In stag- nant water. L. pusilla, Rabh. Length 60 to 70 /*, diameter 0.5 to 0.6 u,. L. parasitica, Ktz. Length 90 to 150 ^, diameter 1 /^. L. radians Ktz., and L. spissa, Rabh. Parasites upon marine algce. CLASSIFICATION OF THE BACTERIA. 91 g. Beggiatoa, Trev. Filaments very slender, surrounded by mucous mat- ter, rigid, having oscillatory movements. Protoplasm white, enclosing numerous granules, which recent observations have demonstrated to be crystalline sul- phur (Cramer, C.ohn). The Beggiatoa are found most abundantly in thermal sulphur waters, where they constitute flocculi, which have been named Glairine, Bare- gine. They often live in water not containing free oxygen. They play a great role in the elimination of sulphur and the disengagement of sulphuretted hydrogen in thermal waters. Their principal species are : — B. alba, Trev. A whitish mucous mass enclosing colorless filaments, having a diameter of 3.5 to 4 //,. In most thermal and stagnant waters. B. arachnoidea, Rabh. Flocculi very minute, snow white, filaments as long as broad, — 5.4 to 7 p. In the thermal waters of Europe. B. nivea, Rabh. ; B. leptomitiformis, Trev., nearly related species, living in the same conditions. Cohn and Warming have also described : — B. mirabilis, Cohn, articles scarcely flexible, measuring 20 to 40 p. B. minima, Warming, a very small species, very flexible ; length 40 /*, thickness 1.8 to 2 /*. 4. SPIEOBACTERIA. This tribe includes the bacteria with undulating filaments, or filaments in spirals, more or less cle- 92 MORPHOLOGY OF THE BACTERIA. veloped, from the Vibrio rugula, which only pre- sents a single curve in its centre, to certain species of Spirillum which have numerous turns of the spiral. In several species, cilia, or a flagellum, have recently been observed. We divide them into three genera : — Fil. short, slightly sinuous . . . VIBRIO. Fil. short, spiral, rigid .... SPIRILLUM. Fil. long, spiral, flexible .... SPIROCH^TE. g. Vibrio, Auct. emend. Body filiform, more or less distinctly articu- lated, always undulating, having serpentine movements. This genus forms the transition between the Desmobacteria and Spirillum"hom which it cannot be separated " (Warming). Fil. thick, with a single curve ... V. RUGULA. Fil. slender, with several undulations . V. SERPENS. V. rugula, Muller (V. lineola, Duj. ex parte). Filament presenting in its centre a single curva- ture, feeble but distinct ; length 8 to 16 p. The shortest are slightly curved (= 6 /A Warming), the larger, which may attain 17.6 /j, (Colin), 35 /& (Warm.), are about to divide. Movements of rotation more or less rapid around their longer axis ; of progression forward, giving then the idea of a serpentine move- ment: having a cilium (Warming). V. rugula is commonly found in swarms, in infusions, in deposits upon the teeth, in intes- tinal matters (Leeuwenhoeck), in choleraic dis- charges (Pouchet). CLASSIFICATION OF THE BACTERIA. 93 V. serpens, Miiller. Filament one half less in diameter than the pre- ceding, rigid, annulate, having two or three regular undulations, at least two in the shortest ; height of one turn of the undulations 8 to 12 ^,, diameter 1 to 3 n, total length 11 to 25 /*, thickness 0.7 p ; move- ments analogous to those of B. subtilis ; having a cil- ium (Warm.). In numerous swarms in infusions, river water, etc. g. SpirochsBte, Ehrb. S. plicatilis, Ehrb. Filament not extensible, twisted in a long helix, flexible, the turns of the spiral near together ; suscep- tible of twisting upon its axis and of an undulatory movement ; total length 130 to 200 p. Rare species; in infusions, stagnant water, sea-water, etc. S. Obermeieri, Cohn. Does not differ from the preceding, either in size, conformation, or in its movements, but by its habitat and physiological peculiarities. In the blood of persons attacked by recurrent fever (Oberaieier, 1872, Weigert, Birch-Hirsch- feld, etc.) during the period of access, never during the remission. S. gigantea. Warming. Found upon the coasts of Den- mark ; thickness of body, 3 /z, height of spiral 25 /u, diam- eter 7 to 9 i. 94 MORPHOLOGY OF THE BACTERIA. g. Spirillum, Ehrb. Filament spiral, rigid; turns of spiral short and regular. S. tenue, Ehrb. Filament slightly tortuous, three to four turns of the spiral ; length and diameter of a single turn, 2 to 3 /u,. When the filament has a turn and a half, it re- sembles an fl ; the filaments of two to five turns have a length of 4 to 15 /* ; spiral movement very rapid. In infusions, etc. S. undula, Ehrb. (Vibrio prolifer, Ehrb.) Filament larger, turns of the spiral wider apart (from 3 to 5 /A) ; having usually one half a turn to one full turn, rarely one and a half, two, or three ; length 8 to 10 p, breadth 5 ^, thickness of filament 1.3 /A ; having a very rapid spiral movement. Fetid animal and vegetable infusions and run- ning water. The S. rufum, Pertz, only differs from this by its reddish color. S. volutans, Ehrb. Filament large and thick, turns of spiral regular, well separated, and 13 p in height ; number of turns two, three, and three and a half, rarely six and seven ; total length 25 to 30 yu,, thickness 1.5 //-, breadth 6.6 //,; movement sometimes rapid, sometimes motionless ; a well-defined cilium, already seen by Ehrenberg (Cohn, Warming). This giant of the bacteria is found in vege- table and animal infusions, in sea-water, and in fresh water. From Micr. Journ. Vol.. XIII, ITS. Pl.V. &$ £• % *r • 6 o hi 5W® Qb -,<,y^^w *v-^-* 11 «» SdJ^f^, S',f!"'V, >*\VI •/^*. Jjlw XWSi'W ' , \'/ V'V'^^ W "^3-- * % % s:SF3y^ «!!!> PLATE V. From " Microscopical Journal'' FIG. 1. — Micrococcus prodigiosus (Monas prodigiosa, Ehr.). Spherical bacteria of the red pigment, aggregated in pairs and in fours ; the other pigment bacteria are not distinguishable with the microscope from this one. FIG. 2. — Micrococcus vaccince. Spherical bacteria, from pock-lymph in a state of growth, aggregated in short four to eight-jointed straight or bent chains, and forming also irregular cell-masses. FIG. 3. — Zoogloea-form of micrococcus, pellicles or mucous strata characterized by granule-like closely set spherules. FIG. 4. — Rosary chain (Torula-form) of Micrococcus urece, from the urine. FIG. 5. — Rosary-chain and yeast-like cell-masses from the white de- posit of a solution of sugar of milk which had become sour. FIG. 6. — Saccharomyces glutinis ( Cryptococcus glutinis, Fersen.), a pullu- lating yeast which forms beautiful rose-colored patches on cooked potatoes. FIG. 7. — Sarcina spec,* from the blood of a healthy man,** from the surface of a hen's egg grown over with Micrococcus luteus, forming yel- low patches. FIG. 8. — Bacterium termo, free motile form. FIG. 9. — Zooglcea-form of Bacterium termo. FIG. 10. — Bacterium-pellicle, formed by rod-shaped bacteria arranged one against the other in a linear fashion, from the surface of sour beer. FIG. 11. — Bacterium lineola, free motile form. FIG. 12. — Zoogloea-form of B. lineola. FIG. 13. — Motile filamentous Bacteria, with a spherical, or elliptical highly refringent " head," perhaps developed from gonidia. FIG. 14. — Bacillus subtilis, short cylinders and longer, very flexible motile filaments, some of which are in process of division. FIG. 15. — Bacillus ulna, single segments and longer threads, some breaking up into segments. FIG. 16. — Vibrio rugula, single or in process of division. FIG. 17. — Vibrio serpens, longer or shorter threads, some dividing into bits, at * two threads entwined. FIG. 18. — " Swarm " of V. serpens, the threads felted. FIG. 19. — Spirillum tenue, single and felted into " swarms." FIG. 20. — Spirillum undula. FIG. 21. — Spirillum volutans* two spirals twisted around one another. FIG. 22. — Spirochcete plicatilis. All the figures were drawn by Dr. Ferdinand Cohn with the immersion lens No. IX. of Hartnack Ocular III., representing a magnifying power of 650 diameters. 96 MORPHOLOGY OF THE BACTERIA. M. Warming has recently described three new species found upon the coast of Denmark : — Sp. violaceum, height 8 to 10 /x, diameter 1 to 1.5 /x, thick- ness 3 to 4 fi ; a ciliuin at each extremity. Sp. Rosenbergii, height of helix 6 to 7.5 /*, thickness 1.5 to 2.6 /x. Sp. attenuatum, body very attenuated at the two extrem- ities, without a cilium. We give below some details concerning the other colorless Schizophytes : — g. Sarcina, Goods. The Sarcina, which it is useless to describe here, can be considered as bacteria in which the division occurs by two perpendicular par- titions in such a manner that multiplication takes place in two directions. Sarcina is very nearly allied to Merismopedia, from which it only differs by the absence of chlorophyll; its siliceous skeleton allies it with the diatoms. g. Ascococcus, Billr. Cells hyaline, small, globular, closely united in globular or oval families, irregularly lobed and lobu- lated, surrounded by a thick gelatinous envelope, cartilaginous, forming a soft membrane, flaky, easily separating. A. Billrothi, Cohn. Families in masses of 20 to 160 p, surrounded by a thick membrane of 15 \i. In a solution of tartaric acid exposed to the air. g. Myconostoc, Cohn. Filaments very slender, colorless, folded, rolled up in a mucous substance, united in very small globules. CLASSIFICATION OF THE BACTERIA. 97 M. gregarium, Cohn. Unique species found on the surface of a putrefying infusion. g. Cladothrixi Cohn. Filaments in form of leptothrix, very slender, color- less, not articulated, rigid or a little undulating, falsely dichotomous. Cl. dichotomy Cohn. In foul water. g. Streptothrix, Cohn. Filaments in form of leptothrix, very slender, color- less, not articulated, straight or slightly spiral, a little branched. Str. Fcersteri, Cohn. In concretions in the lachrymal canal of man. PLATE VL From photo-micrographs made in Havana and New Orleans. Re- produced by permission of the National Board of Health. FIG. 1. — Spiroch&te (plicatilcf). From foul bilge-water, Hav- ana, Sept. 1879. X 1,450 by Zeiss's T\ in. objective. FIG. 2. — Vibrios from water of harbor, Havana, near discharge of sewer, Aug., 1879. X 1,450 diameters by Zeiss's ^ in. objective. FIG. 3. — Sarcina (sp. ?). From standing water in flower-vase, Lafayette cemetery, New Orleans, April, 1880. X 400 diameters by Beck's \ in. objective. FIG. 4. — Spirillum (volutans ?). From foul gutter- water, New Orleans, May, 1880. X 600 diameters. PLATE VI. FIG. i. FIG. 2. FIG. 3. FIG. 4. PART SECOND. PHYSIOLOGY OF THE BACTERIA. CHAPTER I. DEVELOPMENT OF THE BACTERIA. THE bacteria are now known to us from a mor- phological point of view : let us proceed to study the life of these microscopic beings ; first, from a general point of view, that is to say, by study- ing their functions of nutrition and reproduction, independently of the special characters impressed upon these functions by certain media; then by considering the relations which are established between the bacteria and the particular media in which they may be developed. The bacteria are of all beings the most widely diffused. We meet them everywhere, — in the air, in water, upon the surface of solid bodies, in the interior of plants and animals. If we expose a transparent liquid containing traces of organic substances, we find after a short time that it has become clouded, and the microscope shows us that it contains myriads of these beings. What is the source of these organisms so widely disseminated, and which develop so rapidly? This PLATE VII. DISSEMINATION OF THE BACTERIA. From photo-micrographs made in New Orleans. Copied by permission of the National Board of Health. FIG. 1. — Spirillum (Sp.?) from water of salt marsh, near Salem, Mass. X 400 diameters by Beck's | in. objective. FIG. 2. — Bacteria in distilled water (see note on page 107). X 1,000 diameters by Zeiss's -j^ in. objective. FIG. 3. — Leptothrix buccalis, epithelial cell, etc., from human mouth. X 1,000 diameters by Zeiss's -fa in. objective. FIG. 4. — Bacteria from human foeces. X 1,500 diameters by Zeiss's -fa in. objective. PLATE VII. <^ k * \X y v- ' •-•- r r/x ;^ -.1. FIG. i. . FIG. 2. »» .-., iE»l^ % \ • » .1 -^ar Fm. 4. DEVELOPMENT OF THE BACTERIA: 101 is the first question which presents itself, — a ques- tion which has given rise to long discussions, in the examination of which we shall only enter in order to give a short historical statement. § 1. — ORIGIN OF THE BACTERIA. The origin of the bacteria, as of all the other inferior organisms, is conceived in three different manners : — 1. For some, these organisms are produced by heterogenesis ; that is to say, by creation outright from mineral or organic substances (spontaneous generation). 2. According to others, they come directly from individuals like themselves, by one of the known modes of generation, — fission, spores, etc. 3. Finally it is believed that they are derived from organisms already existing, and are nothing more than different states or phases of develop- ment of known species, of which the life cycle is not yet discovered. We will examine the latter hypothesis, which constitutes what is called polymorphism, when we speak of the phenomena of reproduction. As to the two first, we will content ourselves with indicating the late works which have appeared for and against each; insisting above all upon the facts which relate to the proof of the presence of bacteria or their germs in the air, water, and liquids or tissues of the human organism, — - blood, urine, etc. 102 PHYSIOLOGY OF THE BACTERIA. Hetero genesis. — Since the experiments of Pou- chet and of his pupils, and the arguments given by MM. Trecul and Fremy, the last facts invoked in favor of heterogenesis are due to MM. Onimus, Servel, Bastian, etc. M. Onimus contends that the " proto-organisms may be born in media, protected against the air, which contain albuminoid substances." M. Martin sustains an analogous idea. Accord- ing to him, the bacteria are derived from protein granules. According to Neusch, bacteria are pro- duced in the interior of animal or vegetable cells without any lesion and without coming from the air. To demonstrate this he plunges divers fruits under water, in saline or acid liquids (phosphates, sulphates, carbonate of potassa, etc.), and he finds there bacteria; but, according to him, these are not living organisms, properly so called, but ab- normal cellular vegetations. M. Servel, decapitating some guinea-pigs, caused the heads, the livers, and the kidneys to fall into a solution of chromic acid, 1 to 100. At the end of several days, the superficial parts were hard- ened ; but the centre was softened, and filled with bacteria. The presence of bacteria in eggs has several times been verified, and the heterogenists have hastened to draw an argument from this fact in favor of their theory. M. Gayon explains the ap- pearance of these organisms in the eggs of birds by their presence in the normal state in the oviducts. DEVELOPMENT OF THE BACTERIA. 103 Finally, Bastian, having succeeded in obtaining bacteria in liquids which he believes deprived of every germ, believes in their spontaneous genera- tion. The following is a resume of his experiment : Normal acid urine is brought to the boiling- point, then a solution of potash (in sufficient quan- tity to neutralize the volume of urine employed) is also brought to the boiling-point ; after cooling, the two liquids are mixed, and the whole placed in an oven at 50°. At the end of two or three days, bacteria are developed. Pasteur points out three causes of error in the experiment of Bastian : 1. The germs may come from the urine ; 2. The germs may come from the solution of potash ; 3. The germs may be fur- nished by the vessels employed in the experiment. In support of this criticism, Pasteur has made some similar experiments, guarding against these causes of error, and has not obtained bacteria. DISSEMINATION OF BACTERIA IN AIR AND WATER. Air. — The experiment of Pasteur for gathering atmospheric germs is well known. He fixes a glass tube in an aperture made in a window-blind. The extremity of the tube, which communicates with the open air, is closed with a plug of cotton, to the other extremity is attached an aspirator. When the air has filtered through the cotton for some hours, this is examined, and is found to be filled with germs. 104 PHYSIOLOGY OF THE BACTERIA. Before Pasteur, Ehrenberg and G. de Ciaubry had already announced the presence in the air of the eggs of infusoria. Robin had also recognized that the atmosphere contains, in addition to all sorts of debris, spores, pollen-grains, portions of insects, and rarely the eggs of infusoria. More recently Maddox and Cunningham, by the aid of an aeroscope invented by the former, gathered numerous microbes, as well as bacteroid particles. Tyndall, by causing a ray of light to enter a dark- ened chamber, has rendered visible all these mi- nute corpuscles. His researches show that the optical examination of air enables us to determine in an exact manner the presence or absence of germs. Let us also mention the experiments recently made by Miquel in the park of Montsouris. This observer has found in the atmosphere a consider- able number of germs. For the forms of which the diameter exceeds 2 //,, he has ascertained that 66 the average number of microbes in the air is feeble in winter and augments rapidly in spring, etc. ; 2. That rain always diminishes the number of these microbes; 3. That rain-water introduced with the greatest precautions, into flasks with slen- der curved necks, first heated to destroy germs, rarely contains rotifers, etc., but always contains bacteria." En resume, the existence of germs can be dem- onstrated, 1, by direct research ; and 2, by cultiva- tion. Direct research may be made by the optical examination of the air (method of Tyndall), the DEVELOPMENT OF THE BACTERIA. 105 microscopic examination of dust (method followed by Marie-Davy, Tissandier), the examination of particles obtained by filtration, by gathering germs with an aeroscope, by condensation of atmospheric moisture upon refrigerating vases., etc. The culti- vations consist in exposing to the air which is to be examined some liquids in which all pre-existing germs have been destroyed (Pasteur, Tyndall, etc.). This method has shown that liquids exposed in an atmosphere deprived of all germs does not undergo putrefaction, but this occurs as soon as the access of air not deprived of germs is per- mitted (Tyndall). All of these methods give concordant results ; deposits containing germs of various kinds are always obtained. But this objection presents itself to the mind : Do the bacteria obtained by cultiva- tion exist in the atmosphere ? or do they come from germs which have developed rapidly upon finding a favorable medium ? From the experi- ments of Cohn, Miquel, etc., it is known that the atmosphere contains very few adult bacteria. Mi- quel in a recent communication says, in effect, that bacteria are rarely found in the air in a complete state, but rather under the form of shining points, difficult to distinguish directly one species from another. Are not these brilliant points Micrococcif In other terms, the air contains permanent spores, organisms which, as we shall see in speaking of the reproduction of the bacteria, develop at a certain period of the existence of the adult forms, in their interior, which escape from the sporogenous fila- 106 PHYSIOLOGY OF THE BACTEEIA. m en t, are drawn into the air by the evaporation of the liquid containing them, or, after dessication, by the winds. These spores are the point of depart- ure of epidemic foci, and their extreme lightness explains how readily they are disseminated by the winds. Water. — Water contains considerable quantities of bacteria and especially of germs. Their pres- ence in atmospheric water is established by the experiments of Lemaire and Gratiolet, — and after them by more recent observers, — by means of con- densers filled with ice, and placed in the fields and for comparison in closed apartments. Rindfleisch lias since expressed the opinion that the vapor of water does not contain spores or bacteria, and that telluric waters alone contain them ; but Billroth, Colin, and others have proved that Rindfleisch was too positive in his statement. It is not surprising that telluric waters contain such a quantity of bacteria that their existence is admitted by all. The dust gathered upon the sur- face of stones, of leaves, of fruits, etc., shows upon microscopic examination an abundance of germs (Marie-Davy, Tissander) ; the washing of these objects and of the soil by the rain transports them into the rivers and from the rivers to the sea, which contains considerable quantities of them. Thus, a drop of water from the Seine, according to Pasteur and Joubert, is always fecund, and may give birth to several species of bacteria. The dis- tilled water of laboratories also contains germs, and DEVELOPMENT OF THE BACTERIA. 1C7 these of so small a diameter that they pass through all filters.1 Cohn has proved that some are not arrested by a super position of sixteen filters. The only waters which do not contain them are those drawn from the very source of a spring. DISSEMINATION OF BACTERIA IN THE HUMAN ORGANISM. If bacteria are so generally disseminated in the great external media, it is not surprising that they are found on the surface of the human body and in the interior of the organs in communication with the exterior. But to account for their pres- ence in the interior of organs we find ourselves in presence of two hypotheses : one admitting the spontaneous production of these organisms in the interior of the tissues, the second explaining it by the introduction through the membranes of the germs of bacteria from without. 1 Having been directed by the National Board of Health to make some experiments with a view to confirming or disproving the results of Klebs and Crudelli, who claim to have found the germ of malarial fevers in the atmosphere of the Pontine marshes near Rome (their Bacillus nia- larice), I aspirated ten gallons of air on the edge of a swamp in the vicin- ity of New Orleans, through 4 c.c. of distilled water. Upon examining this water with the microscope on the following morning, I was surprised to find a large number of actively moving bacteria and monads ( A/o/w.v lens}. To make sure that these really came from the air, I examined my distilled water, which had been standing in the laboratory for several weeks (in a bottle, corked, but occasionally opened as distilled water was required) and found the s-ame forms present in considerable numbers, not so numerous, however, as in the water through which swamp air had been drawn. As the germs were present in the distilled water, I presume that the passing of air through it for several hours, and the organic matter contained in it, favored the development and multiplication of these micro-organisms. Subsequent experiments with freshly distilled water gave very different results as to the number of organisms found. See fig. 2, plate vii. — G. M. S. 108 PHYSIOLOGY OF THE BACTERIA. In truth, the cutaneous surfaces are penetrated with difficulty by germs, although the hairs upon the surface of the body serve to collect them. The short hairs in the nares prevent, to some ex- tent, the atmospheric germs from penetrating into the bronchi, but this protection is not sufficient ; and, notwithstanding the mucus of the1 nasal fossse and of the pharynx, they may be found in the al- veoli of the lungs, if we may believe Kindfleisch and Eberth. Do the bacteria pass into the blood ? They may be transported in food and drink into the alimentary canal, where an elevated tempera- ture, the presence of saliva, etc., favor their de- velopment. On the other hand, the acid secretions of the stomach, the bile, and the pancreatic juice moderate, if they do not prevent, the multiplica- tion of these organisms. The presence of bacteria in normal blood and urine, or their occasional entrance into these fluids, are important questions, which have induced many contradictory researches, but which are not yet definitely settled.1 1 " If there is any organism in the blood of yellow fever demon- strable by the highest powers of the microscope as at present perfected, the photo-micrographs taken in Havana should show it. No such organ- ism is shown in any preparation photographed immediately after collection. But in certain specimens kept under observation in culture cells, hyphomy- cetous fungi and spherical bacteria made their appearance after an inter- val of from one to seven days. The appearance of these organisms was, however, exceptional ; and in several specimens taken from the same individual at the same time, it occurred that in one or two a certain fun- gus made its appearance, and in others it did not. This fact shows that the method employed cannot be depended upon for the exclusion of atmos- pheric germs, but does not affect the value of the result in the consider- able number of instances in which no development of organisms occurred DEVELOPMENT OF THE BACTERIA. 109 Two kinds of researches have been undertaken for the purpose of discovering germs in normal blood. The direct method, or microscopic exam- ination, has given results very much disputed. The blood contains, indeed, a considerable number of little granules, of which the nature is doubtful, and which it is difficult to distinguish from Micro- coccus. Thus, while Liiders asserts that normal blood contains germs, or spores, which only awrait a favorable alteration in the fluid in order to de- velop themselves, Rindfleisch formally denies their existence. The indirect method, which consists in cultivat- in culture cells in which blood, in a moist state was kept under daily observation for a week or more. " The method employed seemed the only one practicable for obtaining blood from a large number of individuals without inflicting unwarrant- able pain and disturbance upon the sick. It was as follows : One of the patient's fingers was carefully washed with a wet towel (wet sometimes with alcohol and at others with water), and a puncture was made just back of the matrix of the nail with a small triangular-pointed trocar from hypodermic syringe case. As quickly as possible a number of thin glass covers were applied to the drop of blood which flowed. And these were then inverted over shallow cells in clean glass slips, being attached usually by a circle of white zinc cement. In dry preparations, which are most suitable for photography, the small drop of blood was spread upon the thin glass cover by means of the end of a glass slip. " The thin glass covers were taken from a bottle of alcohol, and cleaned immediately before using; and usually the glass slips were heated shortly before applying the covers, for the purpose of destroying any atmospheric germs which might have lodged upon them. These precautions were not, however, sufficient to prevent the inoculation of certain specimens by germs floating in the atmosphere (Penicillium and micrococci) ; and in nearly every specimen the presence of epithelial cells, and occasionally a fibre of cotton or linen, gave evidence that under the circumstances such contamination was unavoidable. It is therefore be- lieved that any organism developing in the blood of yellow-fever, or of other diseases collected by the method described, or by any similar method, can have no great significance, unless it is found to develop as a rule (not occasionally) in the blood of patients suffering from the dis- 110 PHYSIOLOGY OF THE BACTERIA. ing normal blood in flasks perfectly closed, has also given some favorable results, such as those of Hensen, Tiegel, Billroth, and Nedvedsky, and some unfavorable results, as those of Liiders and Pasteur. According to Nedvedsky 7 the blood " con- tains germs capable of undergoing in it, under certain circumstances, an ulterior development: these are the Hemococcos." If these germs do not give birth, normally, to bacteria, it is because the blood is as injurious to them as the most advanced stages of putrefaction (Billroth). If this hypoth- esis is true, it explains several embarrassing facts, such as the existence of micrococci in the pus of ease in question, and is proved by comparative tests not to develop in the blood of healthy individuals, obtained at the same time arid by the same method. " Tried by this test, it must be admitted that certain fungi and groups of micrococci, shown in photographs taken from specimens of yellow- fever blood collected at the military hospital and preserved in culture cells, cannot reasonably be supposed to be peculiar to or to have any causal relation to this disease." — Preliminary Report of Havana Commis- sion to National Board of Health. In subsequent observations upon the blood of malarial fever, of syphilis, and of leprosy, I have sometimes obtained a development of micrococci in culture cells where all possible precautions as to the exclu- sion of atmospheric germs had been taken, and in one case have seen the development of Penicil/ium in another of Sarcina. The last observa- tion is, so far as I know unique, and I have still in my possession the culture-slide containing numerous masses of Sarcinti, presenting the characteristic arrangement of the cells in fours. This slide was put up at the bedside of a patient suffering from intermittent fever in the Char- ity Hospital, New Orleans. Every precaution was taken to exclude at- mospheric germs. The patient's finger was washed with absolute alcohol just before making the puncture from which the little drop of blood was obtained. The question as to whether in this and similar cases the germs of the organism which develops come from the atmosphere or pre-existed in the blood is one to which I propose to give special atten- tion ; and, after further experiment, I shall discuss it in my report to the National Board of Health. — G. M. S. DEVELOPMENT OF THE BACTERIA. Ill closed abcesses, in cysts, in urine drawn from the bladder, etc. § 2. — NUTRITION AND KESPIRATION OF THE BACTERIA. The bacteria, being organisms composed of a cell membrane of cellulose, and of protoplasmic contents, deprived of chlorophyll, must receive nutriment and respire in the same manner as all the colorless vegetables and all the inferior animals deprived of special apparatus, — that is to say, by end osmotic absorption. Although the media in which the bacteria de- velop are various, yet, from the point of view of the nutritive function, they act everywhere ac- cording to the same laws. No matter in what medium they live, they must have water, nitro- gen, carbon, and oxygen, as well as certain min- eral salts which enter, but in quantities exceedingly minute, into the chemical constitution of all organ- ized bodies. Water. — This element is indispensable to the active life and development of the bacteria. Dessi- cation arrests completely the movements of those which are mobile, and the functions of all the bacteria in general ; but it does not kill them, at least if it be not prolonged beyond a certain time. The Micrococci of different kinds of virus are examples of the continued vitality of these organisms after dessication for a considerable time. 112 PHYSIOLOGY OF THE BACTERIA. The bacteria present in this respect numerous va- riations according to the species and the period of development which they have attained. In the state of permanent spores, they are extremely ten- acious of vitality. They resist for a long time not only dessication, but a considerable elevation of temperature. Among the bacteria, some are developed in liq- uids, — the greater number, — others upon damp surfaces. The former can live in fresh water, sea- water, thermal waters, and the liquids of animal or vegetable organisms, etc. A surprising fact is, that the composition, so different, of fresh and sea water appears to have no influence upon the bacteria. We find in both all the species, from Bacterium termo to Spirillum volutans. Nitrogen. — Pasteur has demonstrated that it is not necessary that the nitrogen which is to serve as nutriment to the bacteria should be in the form of albumen, but that these organisms can take posses- sion of it in the form of ammonia. In fact, in Pasteur's solution, composed as fol- lows : — Distilled water 100. Sugar candy 10. Tartrate of ammonia .... 1. Ashes of one gramme of yeast . 0.075. the bacteria increase sometimes with such rapidity that they interfere with the development of the alcoholic ferment. DEVELOPMENT OF THE BACTERIA. 113 Cohn, in order to better observe the phenomena and to get rid of the moulds, which the cane-sugar caused to develop too rapidly, employed the fol- lowing culture-fluid : — Distilled water .... 100. Tar tr ate of ammonia . . 1. Ashes of yeast .... 1. Bacteria develop in this fluid wonderfully, which proves that sugar is not indispensable to them. One other solution often employed is that of Mayer. It has the advantage of not requiring the employment of ashes of yeast : — Phosphate of potash . . .0.1 gramme. Sulphate of magnesia . . . 0.1 „ Tribasic phosphate of lime . 0.1 „ Distilled water 20 c.c. Cohn adds to this 0.2 gr. tartrate of ammonia. En resume, the bacteria can take nitrogen, which they need in order to form their protoplasm, either from albuminous compounds, which they decom- pose, as in putrefaction, or in the form of am- monia, or, perhaps, by borrowing it from nitric acid, but this last source is not well established (Cohn). Carbon. — In addition to the sources common to other organisms, the bacteria can take this im- portant element of their composition, under cer- tain circumstances, from the organic acids. Thus, when we cultivate bacteria in a solution containing 8 114 PHYSIOLOGY OF THE BACTERIA. only tartrate of ammonia with a small quantity of mineral salts (phosphoric acid, potash, sulphuric acid, lime, and magnesia), they develop rapidly, taking their carbon from the tartaric acid. Cohn has endeavored to ascertain if other or- ganic acids could be assimilated by the bacteria. By making use of succinate of ammonia, or neutral acetate of ammonia, he has been able to cultivate these microphytes. Besides, as Pasteur had already experimented with solutions containing lactates, and in which bacteria had developed until the salt had completely disappeared, we may admit that the bacteria can assimilate the organic acids, — tartaric, succinic, acetic, and lactic ; but tartaric acid seems to furnish the best alimentary solution. Other substances containing carbon are also as- similated by the bacteria, — cane-sugar, milk-sugar, glycerine, and even cellulose (according to Mit- scherlich). Cohn concludes, " that the bacteria multiply quite normally, and in great quantity, whenever they find the elements in solution which constitute ashes, and that they can take the carbon which they need from any organic substance containing it, and their nitrogen from ammonia, urea, and probably from nitric acid. The bacteria, then, re- semble green plants, in that they assimilate nitro- gen contained in their cells by taking it from ammonia compounds, which animals cannot do. They differ from green plants in that they cannot draw their carbon from carbonic acid, and only assimilate organic substances containing carbon, DEVELOPMENT OF THE BACTERIA. 115 above all the hydrates of carbon and their deriv- atives ; and in this respect they resemble animals." Absorption. — How are these various substances absorbed ? The observations of Grimm, Hoffmann, de Seynes, etc., permit us to assure ourselves that these organisms absorb by endosmosis the sub- stances upon which they are nourished. Grimm, upon examining with the microscope some particles of lemon containing bacteria and spores of algse, saw a certain number of the former gather around a spore, and fix themselves to it by one of their extremities. They did not pene- trate it ; but when they abandoned it, the spore had diminished in volume, and lost a portion of its contents, while the bacteria had taken a greenish color. Hoffmann has seen that these little organisms, when placed in a solution of carmine or of fu- schine, after a time are colored an intense red, while the mucus surrounding them remains color- less. De Seynes, also, from his observations upon the vibrios which accompanied some colored fila- ments of Penicittium glaucum, believes that bacte- ria are susceptible of absorbing coloring matters from vegetables and from animals with which they are in contact. Oxygen. — The role of oxygen in the life of the bacteria has given rise to numerous controversies. First, it seems a priori that the bacteria ought 116 PHYSIOLOGY OF THE BACTERIA. to act like all other living beings, and to respire like the other inferior organisms deprived of chlo- rophyll — that is to say by absorbing oxygen and eliminating carbonic acid. This is, indeed, the opinion of a great number of botanists. But, according to Pasteur, it is not so with the bacteria. When we examine what occurs in putrefaction, we find that at first certain species are developed (Monas crepusculum, Bacterium termo, etc.), which absorb all the oxygen dissolved in the liquid, and come to the surface where they form a thick veil ; after this, other species of vibrioniens appear, which are developed in a medium entirely de- prived of free oxygen, by borrowing this gas from the fermentable matters contained in the liquid. These chemical decompositions constitute putrefaction. The first of these organisms, regarding the na- ture of which Pasteur has long been uncertain, are aerobies : they live in contact with the air, and have need of oxygen. The second, anaerobiez, not only have no need of oxygen, but are killed by it. These differences in the respiration of organ- isms belonging to the same group are not admitted by a great number of recent observers. Hoff- mann, among others, says expressly : " These little beings cannot live without air, I should say with- out oxygen : if this gas is wanting, they cease to move and do not multiply at all. If a drop of liquid full of bacteria is placed upon a glass slip, then covered by a piece of thin glass, the active DEVELOPMENT OF THE BACTERIA. 117 bacteria will all approach gradually to the margins of the cover ; and it is there that at the end of several days, after the successive death of the greater number, some are still found endowed with life and movement. If a similar preparation is at the same time protected by an impermeable ce- ment against dessication and against the introduc- tion of atmospheric air, all movement among the bacteria will cease at the end of two minutes, pro- vided, however, that no air bubble has been im- prisoned with the liquid." The influence of oxygen upon the life and de- velopment of bacteria is also very manifest in an experiment recently made, and not yet published, by Toussaint, who has been kind enough to com- municate it to me. In studying the development of the spores of Bacillus anthracis in the moist chamber of Ranvier, Toussaint has observed the following curious facts, which offer a striking analogy to those above mentioned, borrowed from Hoff- mann. " The bacteria, which occupy the cen- tral portion of the moist chamber and which by reason of their situation receive very little oxygen from the groove, are soon arrested in their development ; while those which occupy the borders are long and heaped up in immense num- bers, those in the centre remain small, formed of two, four, or five articles, which are easily sepa- rated from each other; they soon cease to grow and are not transformed into spores." Cohn is also as explicit. " There is no doubt," 118 PHYSIOLOGY OF THE BACTERIA. he says, " that the complete development of Bacil- lus, and above all reproduction by means of spores, is only made under the influence of free access of air." We might explain the contradictory facts of Pasteur by admitting, with Cohn, that the appear- ance of different roles played by the aerobics (Bacterium) and the anaerobies (Bacillus) is sim- ply due to a veritable struggle for existence which takes place between the microbacteria and the desmobacteria. ACTION OF VARIOUS AGENTS UPON THE BACTERIA. In this paragraph I shall pass in review the action of temperature, of movement, and of va- rious antiseptics. Temperature. — It is very important to study the manner in which bacteria comport themselves under extreme variations of temperature. It is, indeed, upon the results furnished by these re- searches that a great part of the arguments op- posed to the panspermatists by the heterogenists are based. We shall consider the influence upon bacteria of moderate temperatures and of extremes above and below zero. Moderate temperatures — that is to say those which are comprised between 25 and 40° (77 to 104° Fah.) — are generally favorable. The most favorable has been found to be 35° (95° Fah.) (Onimus). DEVELOPMENT OF THE BACTERIA. 119 The degree of resistance to extreme tempera- tures is very variable, according to the species. Thus, according to Frisch, a temperature of 45 to 50° (113 to 122° Fah.) is sufficient to kill B. termo, whilst 80° (176° Fah.) does not kill the " Eacttri- dies " (Bacillus). The permanent spores are especially remarkable by the tolerance which they possess for high tem- peratures. They have been subjected to 100° (212° Fah.) (Schwann), 110° (Pasteur) and even 130° (Schrader) without losing their power of germinating. We must, however, recognize that the results of the experimenters offer the greatest diversity, the result, according to Cohn, of the difficulty of obtaining an equable distribution of the heat in the media, which are generally bad conductors. Cohn has arrived at the following conclusions as the result of numerous experiments made upon the Bacillus of hay infusions : — 1. At a temperature of 45 to 50° (113 to 122° Fah.) the Bacillus still multiplies rapidly, and forms as usual membranes and spores, while the other schizophytes existing in the infusion of hay are at this temperature incapable of multi- plication. 2. At a temperature of 50 to 55° (122 to 131° Fah.) all reproduction and development of Bacillus ceases. It neither forms pellicles or spores; the filaments are 'killed, the spores, on the contrary, preserve, for a longer time (for at least seventeen hours) the property of germinating. 120 PHYSIOLOGY OF THE BACTERIA. 3. While infusions of hay are generally sterilized by a temperature of 60° (140° Fah.) or more, pro- longed during twenty-four hours, certain spores of Bacillus seem able to endure a temperature of 70 to 80° (158 to 176° Fah.) during three or four days without losing the power of germinating. By some experiments made with refrigerating mixtures, Cohn has ascertained that the bacteria are not killed by very low temperatures, acting even during several hours, — 18° for example (0° Fah.). But they are benumbed at a tempera- ture of 0° (32° Fah.) and probably at a temperature a little higher, losing the power of movement and of reproduction, and consequently their action as ferments. They preserve, however, their capacity to resume their activity at a more elevated tem- perature. Frisch has pushed the experiment still further than Cohn. By the evaporation of carbonic acid, he has produced as low a temperature as — 87° ( — 123° Fah.) in liquids containing bacteria, with- out destroying the vitality of these organisms, development having subsequently occurred of coc- cos and of bacteria. Congelation, then, cannot serve to destroy the organized ferments. Let us add, however, that if the passage to ex- treme temperatures is too sudden, there is then an alteration (destruction ?) of these organisms (Schu- macher). Movement. — We would not have consecrated a paragraph to the action of movement upon DEVELOPMENT OF THE BACTERIA. 121 bacteria, if Crova had not recently asserted that movements impressed upon a liquid containing bacteria completely arrests their development. This is an assertion in complete opposition to all that we know of the physiology of these organ- isms, and which it is difficult to reconcile with the fact that bacteria may develop even in the torrent of the circulation. Compressed Air. — We have just seen the in- fluence of air, and especially of oxygen, upon the bacteria. When this agent is in a certain state of tension, it acts in a different manner. M. Paul Bert has proved that under a tension of twenty- three to twenty-four atmospheres all the putrefac- tive processes depending upon the development of vibrios cease to occur. Since, the same savant has found that the anatomical elements and even the red blood globules are killed by oxygen. These researches agree well enough with those of Grossmann and Mayerhauser upon the life of bacteria in gas. From their numerous experi- ments it appears that, under the influence of oxy- gen, there is an exaggeration of the activity of the bacteria ; but if the oxygen is under a pres- sure of five to seven atmospheres, the bacteria live from six to twenty hours, then die, and cannot be resuscitated by atmospheric air. Ozone causes a definite and almost instantaneous arrest of movement. 122 PHYSIOLOGY OF THE BACTERIA. Other gases studied by the same savants have given the following results : — Hydrogen at first causes an acceleration of movement, which is maintained for several days ; then movement becomes less active, and finally it ceases altogether. Carbonic Acid. — Contrary to the facts stated by Pasteur, this agent was found to paralyze the bacteria, and reduced them to complete immobility. If the carbonic acid is displaced by oxygen, the bacteria resume their activity. Chloroform. — This substance, according to the researches of Miintz, arrests the vital phenomena of organized ferments. Miintz uses this charac- ter in order to recognize the soluble ferments, upon which it has no action. JSoracic Acid. — Since the labors of Dumas, which have demonstrated that boracic acid kills the inferior organisms by depriving them of their oxygen, this substance has been employed in vari- ous circumstances as an antiseptic. Sulphate of Quinine. — The action of quinine, either in the state of chlorhydrate or of sulphate, is not yet well established. The experiments of Binz, Manassein, Kroevitsch, Bochefontaine, etc., have, in truth, given contradictory results. Carbolic Acid. — The experiments of Manas- sein have demonstrated that ^th per cent of car- DEVELOPMENT OF THE BACTERIA. 123 bolic acid is sufficient to prevent all development of living beings. It is employed with success in anthrax, in the treatment of wounds, etc. § 3. — REPRODUCTION OF THE BACTERIA. It is well established that the bacteria can mul- tiply by fission, and reproduce themselves also by the formation of endogenous spores. Fission. — The multiplication by fission consists in a transverse division of the cell. When a bac- terium has attained nearly double its ordinary length, we see, in the larger species, that the proto- plasm becomes clearer in the central portion, and a partition forms in the median line separating the contained protoplasm into two portions. The par- tition, at first very delicate, becomes thicker, di- vides into two, and the two articles separate. This phenomenon is produced more or less quickly according to the nature of the medium, its richness in nutritive material, the temperature, etc. When the growth is rapid, the new cells form more quickly than they separate, and are arranged in chaplets. Very often we only find them in this form, in strings of two to four cells coupled together. In some forms the transverse division is preceded by constriction near the middle of the cell. Before the two new cells are separated, the bacterium in this case presents the appearance of a figure 8, and seems to be a simple cell swollen at the two extremities. 124 PHYSIOLOGY OF THE BACTERIA. Under other circumstances, and probably in con- sequence of a mucus transformation of the walls of the mother cells, the new bacteria are envel- oped by a mass of glutinous substance. We have described these masses under the name of Zo- oylcea. The conditions which favor multiplication by fission are, a certain degree of temperature and a sufficient quantity of nutritive material. The higher the temperature, the more rapid is the segmentation of the bacteria, the more rapid their multiplication, — that is to say, up to a certain limit, variable with the species and beyond which the bacteria are destroyed. The multiplication decreases when the tempera- ture is lower, and ceases entirely in the vicinity of 0° (32° Fah.). The influence of richness of nutriment is well seen in artificial cultivation. So long as the bacte- ria find the necessary aliment, in sufficient quantity, to form new protoplasm, they multiply with ac- tivity ; but as soon as the organic matter is de- voured, they cease to divide, fall to the bottom of the vessel, where they accumulate, motionless, and form a deposit more or less abundant. The multiplication of the bacteria by binary fis- sion has for result, if nothing occurs to interfere with the most favorable conditions, the invasion of the medium by an incredible number of these little beings, of which we can only form an idea by calculation. " Let us suppose/* says Cohn, " that a bacterium DEVELOPMENT OF THE BACTERIA. 125 divides into two in the space of an hour, then in four at the end of a second hour, then in eight at the end of three hours, in twenty-four hours the number will already amount to more than six- teen millions and a half (16,777,220); at the end of two days this bacterium will have multiplied to the incredible number of 281,500,000,000; at the end of three days it will have furnished forty- seven trillions ; at the end of about a week, a number which can only be represented by fifty-one figures. " In order to render these numbers more com- prehensible, let us seek the volume and the weight which may result from the multiplication of a single bacterium. The individuals of the most common species of rod-bacteria present the form of a short cylinder having a diameter of a thou- sandth of a millimeter, and in the vicinity of one five hundredth of a millimetre in length. Let us rep- resent to ourselves a cubic measure of a millimetre. This measure would contain, according to what we have just said, 633,000,000 of rod-bacteria with- out leaving any empty space. Now, at the end of twenty-four hours the bacteria coming from a single rod would occupy the fortieth part of a cubic millimeter ; but at the end of the follow- ing day they would fill a space equal to 442,570 of these cubes, or about a half a litre. Let .us admit that the space occupied by the sea is equal to two-thirds of the terrestrial surface, and that its mean depth is a mile, the capacity of the ocean will.be 928,000,000 of cubic miles. The multipli- 126 PHYSIOLOGY OF THE BACTERIA. cation being continued with the same conditions, the bacteria issuing from a single germ would fill the ocean in five days." Reproduction by Spores. — The multiplication by fission, known to the earliest microscopists, has been until recently the only mode of propagation admitted by the authors. Thus M. de Lanessan, in the excellent article which he has devoted to the bacteria, says that the marvellous resources of modern science have not yet permitted us to rec- ognize any other mode of propagation for these organisms. However, M. Ch. Eobin had already, in 1853, indicated the presence in Leptothrix buccalis of little round bodies, " which are perhaps spores." Pasteur has since, in 1865, recognized that " the vibrios of putrefaction and of butyric fermentation present a sort of ovule, or ovoid corpuscle, which refracts light strongly, either in the extremity or in the body of the articles." Later, the same savant, more explicitly, says clearly that these or- ganisms have two modes of reproduction, — by fission and by interior spores (" noyaux "). Towards the same epoch, Hoffmann also pointed out a reproduction by free cellular formation in some bacteria. But we must come to the labors of Cohn, Billroth, and Koch, in order to find pre- cise observations in this regard. The formation of spores has been observed in Bacillus subtilis by Cohn, Bacillus anthracis by Koch, and in Bacillus Amylobacter by Van Tieghem. PLATE VIII. wmtm ' « o FIG. 2. A -r- \ • ° * FIG. 3, Fir,. 4. PLATE VIII. FORMATION OF SPORES IN BACILLUS. From photo-micrographs made in Havana and in New Orleans. Re- produced by permission of the National Board of Health. FIGS. 1 and 2. — Bacillus (ulna ?) found in blood of yellow- fever patient (post mortem) five days after collection. X 3,000 diam- eters by Zeiss's T^ in. objective. FIG. 1. — Rods joined in leptothrix chain. FIG. 2. — A single rod showing spore at one extremity. FIG. 3. — Spores of Bacillus developed in rotten potato, New Orleans, April, 1880. X 1,500 by Zeiss's -^ in. objective. The large cells are some species of Saccharomycete, which was also pres- ent in the same specimen. j?IG. 4. — Development of bacilli from spores, from culture ex- periment with fish gelatine solution. X 1,500 diameters by Zeiss's iV in. objective. 128 PHYSIOLOGY OF THE BACTERIA. Colin, who had in his first publications refused to the bacteria the property of reproduction by spores, thinking that the facts observed by Hoff- mann related to different beings, has verified the experiments of Koch upon the development of B. anthrads, and has himself demonstrated sim- ilar .phenomena in B. subtilis. In culture experiments made with infusion of hay, we see, at a certain moment, in the homo- geneous filaments of the Bacilli very refractive corpuscles making their appearance. Each of them becomes a spore, oblong or in the form of a short filament, highly refractive, and with well-defined outlines. We find the spores ar- ranged in a simple series in the filaments. As soon as the formation of spores has terminated, the filaments can generally no longer be distin- guished, and one would say that the spores were completely free in the mucus ; but their linear arrangement shows always that they are produced in the interior of filaments. Little by little these dissolve, being reduced to a fine powder ; and the spores fall to the bottom of the liquid, where they are found in abundance. The germination of the spore does not seem to occur in the same medium ; but if we take a spore from the deposit formed in an infusion of boiled hay, and transport it into a new infusion, we see the spore swell up, and a short tube form itself at one of its extremities : at this moment it resembles a bacterium with a head. Soon the very refractive body disappears, the tube stretches out into a short rod of Bacillus, com- DEVELOPMENT OF THE BACTERIA. 129 mences to move, and becomes jointed by trans- verse division. Koch, in cultivating the bacteria of charbon in aqueous humor from the eye of the ox, has ob- served some facts exactly similar, both as to pro- duction of spores in linear series in the filaments of Bacillus anthracis and as to the germination of the spore and the birth of a new rod. According to Van Tieghem, the development of Amylobacter is as follows : " The development of a Bacillus includes four successive periods. In the first, the body, cylindrical and slender, recently developed from a spore, stretches out rapidly, and is partitioned ; the articles soon separate (B. subtilis), or remain united in long filaments (B. anthracis). This is the stage of growth and multiplication, two things which at bottom are but one. "Secondly, the articles previously formed, having ceased to elongate and divide, increase sensibly in magnitude, becoming the seat of interior chemical transformations ; and this increase in size operates according to circumstances, in three different man- ners, with some intermediate forms. Sometimes it occurs uniformly throughout the length of the article, which remains cylindrical ; sometimes it is localized, either at one extremity, which is swollen like a tadpole, or in the middle of the article, which swells to a spindle shape. This is the stage of enlargement, or of nutrition, solitary and si- multaneous, which prepares the following state. "In the third period or phase of reproduction 130 PHYSIOLOGY OF THE BACTERIA. there is formed in each article so nourished a spherical or ovoid spore, homogeneous, highly refractive, having a dark outline. At the same time, the protoplasm which occupies the rest of the cavity disappears little by little, and is re- placed by a hyaline liquid, which separates the spore from the membrane ; this dissolves in its turn, and finally the .spore is set at liberty. If the article is swollen in tadpole shape, it is in the ter- minal swelling that the spore has birth ; if it is spindle-shaped, it is near the middle ; if it is cylin- drical, it may be at any point whatever, but is usually near one extremity. The spore when set free germinates under favorable circumstances. At a point where its circumference becomes pale, it gives out a little tube slightly more slender than itself, which elongates rapidly and divides. This fourth period of development or germinative phase brings us back to our point of departure." Sporangia. — Finally, not only do the bacteria develop spores in the interior of their filaments, slightly modified in form, but we may also observe the formation of a veritable sporangium contain- ing many spores. The unpublished observations of M. Touissant, Professor of Physiology in the Veterinary School of Toulouse, give this result, which he has kindly communicated to me. In cultivating spores of the bacteria of charbon in the serum of the blood of the dog, under the microscope, in the warm chamber of Ranvier, Toussaint has seen the filaments take a transverse DEVELOPMENT OF THE BACTERIA. 131 diameter almost double the ordinary diameter, then the protoplasm of the filament to gather together at certain points, — a fact clearly made out, as in the parts where the protoplasm was wanting the bacteria had lost all refractive power. Finally, at a later period the points occupied by the condensed protoplasm augment considerably in volume, and form some ovoid organs more or less elongated, or swollen into a ball, or in the form of a gourd at one extremity. In the interior of these sporangia, from three to six spores afterward form, clearly defined and highly refractive ; then, finally, by breaking up of the membranous enve- lope the spores become free. Toussaint has also followed in the same appar- atus — moist and warm chamber of Ranvier — the mode of germination of the spores. The follow- ing are the most important facts : — The spores are at first highly refractive and animated by brownien movements; at the end of half an hour to an hour, at a temperature of 37 to 40°, in urine, aqueous humor, or serum, the spores lose their refractive power, and their brown- ien movements cease almost entirely; then the spore assumes an aspect slightly granular, it be- comes elongated in the direction of its greatest diameter (they are oval). After two hours of culti- vation, the bacterium has two or three times the dimensions of the primitive spore ; the elongation makes rapid progress, and four to six hours from the commencement of the cultivation, some may 132 PHYSIOLOGY OF THE BACTERIA. be found to occupy the entire field of the micro- scope. From this moment the phenomena which occur differ according to the conditions in which the bacteria are placed. Upon the edge of the air-groove in the moist chamber, the bacteria de- velop very rapidly, forming an interlaced mass ; and in sixteen to eighteen hours, spores may be seen to appear in their interior, — above all, if the preparation has been exposed to light. Often, in this case, the transverse partitions of the filament cannot be seen. If, on the contrary, the bacterium has not been exposed to light, the spores are a longer time in showing themselves (ten or twelve hours more), and almost always division of the filament precedes their formation. In that case, a spore usually appears at each end of the seg- ment in such a manner that the spores belonging to two successive segments are nearer to each other than those in the same segment. Often, also, a spore aborts in a segment (Toussaint). We have seen above, in speaking of the res- piration of bacteria, that the same observer has noted in the course of his experiments some phe- nomena proving the evident influence of oxygen upon the development of Bacillus. It is the same for the formation of spores. And upon this point Toussaint makes the very just remark that the phenomena occur in a different manner in culture experiments and in the human organism. In char- bon, the bacteria never form spores. They remain always relatively short, even in the points where thev form extra- vascular masses, and where conse- DEVELOPMENT OF THE BACTERIA. 133 quently we cannot invoke the movements of the liquid in order to explain their division. The bacteria of charbon, then, take but little oxygen from the tissues : they do not vegetate luxuriantly in the organism; and certainly, if we judge by a calculation necessarily approximative, their devel- opment is seven or eight times less rapid than in the strongly oxygenated serum of culture experi- ments (Toussaint). Polymorphism. — The spores of which we have traced the genesis constitute those germs of which the origin has for a long time been misunder- stood,— those permanent spores or durable spores (Dauersporen), thus called because of their re- markable degree of resistance to temperature, desiccation, and all the agents which kill adult bacteria or arrest their development. These " organs " are disseminated in great num- bers in various media under the form of little rounded corpuscles absolutely similar to the micro- cocci from which it is absolutely impossible to differentiate them. It is, indeed, very probable that the greater part, if not all of these organisms, are the spores of filiform bacteria. In the impossibility of recognizing these forms so nearly related, of referring them to such or such a determined organism, the attempt has been made to cultivate them, in order to follow their development. We have just seen the results of this cultivation for the Bacillus ; but, in the hands of the greater number of experimenters, the re- 134 PHYSIOLOGY OF THE BACTERIA. suits of such culture experiments are far from being so certain. Not having succeeded in re- moving them completely from the invasion of for- eign germs, the greater number have seen the most diverse forms develop themselves, and from this have inferred the most remarkable transfor- mations. Thus, Hallier pretends to have observed the transformation of Micrococcus into various fungi, such as Mucors, Ustilago, etc. The M. of vaccinia comes from Torula rufescens, which is itself a phase of development of Ustilago carbo ; the M. of human variola is derived from a fungus having sporangia and pycnidia, related to Stemphylium sporidesmium ; that of the variola of animals from Cladosporium (Pleospora) herbarum ; the M. of the blood of scarlatina belongs to the g. Tilletia ; those of glanders and of syphilis from a Coniothecium, etc. In the same way Letz- erich has referred the M. of diphtheria to another Tilletia, the T. diphtherica. The transformation of bacteria into " levures " (yeast fungi), and these into Penicillium, has been admitted by Hallier, Trecul, and others. But the researches of Brefeld and de Seynes have shown us that this is far from being demonstrated ; in- deed, in his numerous cultivations, de Seynes has never been able to verify such an affiliation ; and Nageli in his turn has never been able to obtain a transformation of schizomycetes into budding fungi. It is the same as regards the transformation of DEVELOPMENT OF THE BACTERIA. 135 bacteria into moulds and mildews. In some recent cultivations of moulds, made with care, Nageli has never observed the formation of schizomycetes, and reciprocally. Are we not permitted to be- lieve, now that we know of the formation of sporangia among the bacteria, that the micro- scopists who sustain a polymorphism so extended, have taken these organs, of which they have not been able to follow exactly the development, for the sporangia of Mucorini ? This explanation is the more admissible as Trecul has seen the bac- teria " swell up, and transform themselves sepa- rately," a phenomenon quite identical to that -ob- served by Toussaint. En resume. The only change of form well demonstrated in the present state of science, and the only one which can be compared to natural polymorphism, such as it exists in a great number of fungi, consists in the transformation of spores into Bacteria, Bacteridia, Vibrios, etc., and in the different modes of grouping that the cells of bac- teria take in becoming zooglcea, mycoderma, lepto- thrix, etc. To go further would be to lack pru- dence and scientific criticism. 136 PHYSIOLOGY OF THE BACTERIA. CHAPTER II. DEVELOPMENT OF THE BACTERIA IN DIFFERENT MEDIA. IN studying the conditions of life and of develop- ment of bacteria in the different media, natural and artificial, in which they are met, we will con- sider the actions which they determine (or that they accompany) as particular cases of their nutri- tion and of their reproduction. We will con- stantly take, then, their normal physiology as our point of departure ; and we will try to explain in this way the phenomena, so diverse, with which they are associated, — fermentations, putrefactions, con- tagion of infectious maladies, etc. It is especially interesting to study the role of bacteria in non-nitrogenized chemical media, where they accompany the phenomena called fermenta- tion, properly so called ; in nitrogenized media, vegetable or animal, which they transform, as a result of special fermentations, which constitute putrefaction ; in the human organism, where they accompany frequently, if not always, the develop- ment of certain affections having special charac- ters. This will be the object of so many para- graphs. THE BACTERIA IN DIFFERENT MEDIA. 137 § 1. — ROLE OF BACTERIA IN FERMENTATIONS. We say that a liquid is fermenting whenever modifications occur in its chemical constitution, as a result of the nutrition of organized beings. Two kinds of fermentation are commonly distin- guished. In the first group (false fermentations) are arranged those which are produced by soluble quarternary substances (diastase, soluble ferments) secreted by living cells, from which they may be separated in order to study their action upon fer- mentable liquids. This action is comparable to that of certain mineral acids, which operate in the same manner, either by the breaking up of molecules with addition of water or by the phenomena of hydratlon. Veritable chemical reagents, when these substances are once precipitated from their solutions, purified and dried, they preserve their properties indefinitely. A sufficient elevation of tem- perature seems to destroy the edifice of their mol- ecule ; for they lose all their specific power after having been subjected to a temperature more or less elevated, but always inferior to 100° (212° Fah.). In the second group (true fermentations) are joined all the phenomena of chemical modifica- tion which appear intimately united to the devel- opment of inferior organisms, — algse or fungi (figured ferments). Compressed oxygen by kill- ing these ferments, and chloroform by suspending their vital functions, arrest the progress of these fermentations, while the same agents do not mod- 138 PHYSIOLOGY OF THE BACTERIA. ify at all the action of soluble ferments. Accord- ing to Dumas, borax has, on the contrary, the property of entirely destroying the activity of soluble ferments without absolutely preventing certain true fermentations, — for example, the al- coholic fermentation of glucose. We will see fur- ther on that this property of borax has been utilized in the treatment of catarrh of the blad- der and of certain virulent affections. Although at first view these two groups of phe- nomena seem very different, they may, however, be compared the one with the other. Without speaking of the ammoniacal fermentation of urine, which, as we shall shortly see, may be arranged in either of these groups, we may admit that the only difference between these two series of chemical modifications consists in the fact that in one case the true fermentations being the last term in the interior nutrition of the cell have their seat in the interior of the cell itself ; while in the other the first terms of nutrition are always extra-cellu- lar phenomena, having for effect, as Cl. Bernard has shown, to render assimilable or diffusible in the interior of the organism the aliment necessary to the development of every organized being (trans- formation of starch into glucose, of sugar into glucose, emulsion of fats, liquefaction of albumi- noid substances). The study, from a chemical point of view, of these phenomena of nutrition, of these fermenta- tions, since such is their name, has not yet made much progress, and it would be difficult to make a rational classification of them in the present state THE BACTERIA IN DIFFERENT MEDIA. 139 of our knowledge. I will not then seek to clas- sify them, but will content myself with describ- ing them successively, commencing with the best known. I shall only speak of the fermentations caused by the development of bacteria, leaving, consequently, the fermentation which has been best studied, — the alcoholic. I adopt the follow- ing order : — 1. Acetic fermentation of alcohol. 2. Ammoniacal fermentation of urine. 3. Lactic, viscous, and butyric fermentations of sugar. 4. Putrefaction, or nitrification. Acetic fermentation. — The transformation of wine into vinegar is a phenomenon long known and utilized. From a chemical point of view, this transformation is due to oxydation of the alcohol. The following formula represents this reaction : — C2H6O + O2 = C2H4O2 + H20. The agent of this oxydation is a micro-organism called Mycoderma aceti. It belongs to the group of the microbacteria, and we have already given the botanical description of it (page 83) ; but its development presents some interesting peculiar- ities which we think it proper to indicate in the language of M. Duclaux : — " These little beings reproduce themselves with such rapidity that by placing an imperceptible germ upon the surface of a liquid contained in a vat having a surface of one square metre, we may see it covered, in from twenty-four to forty-eight hours, with a uniform velvety veil. If we suppose 140 PHYSIOLOGY OF THE BACTERIA. that there are three thousand cells in a square mil- limetre, which is below the truth, this will give for the vat three hundred milliards of cells pro- duced in a very short time." " The Mycodermi aceti is not always the same. Usually it forms upon the surface of a liquid a soft-looking veil, smooth at first, then wrinkled, which is with difficulty submerged and moistened. If a glass rod is plunged into the liquid, it pierces this veil ; and when it is withdrawn, a portion re- mains attached to the rod ; a*nd the opening made immediately disappears, being occupied by the veil which seems never to have room enough in which to extend itself. In some unpublished experi- ments I have frequently observed another form of veil, dryer, finer, and sometimes showing prismatic colors. This veil does not wrinkle, but is covered with crossed undulations, having sharp edges, which recall the surface of a honeycomb. Sowed upon the surface of various liquids, it reproduces itself identically, and it is difficult not to consider it a different form of the preceding. Finally, I have also met a species of mycoderma producing well-developed veils, but having scarcely any acet- ifying power, and reproducing itself with this character." " It is difficult to distinguish these forms the one from the other, by the microscope, because of their minuteness. We may, however, say that the second which I have described is sensibly smaller than the first, and the third more attenuated than either of the others. However, the differences are feeble." This veil is called the mother of vinegar. The THE BACTERIA IN DIFFERENT MEDIA. 141 liquid in which this mycoderma is cultivated should be a little acid, containing one-half per cent of acetic acid, for example. Under these conditions the Mycoderma vim (a species of Saccharomycete), the formation of which should be avoided, finds conditions unfavorable to its existence. Indeed, this second organism, commonly called flowers of wine, has an action quite different from that of the Mycoderma aceti. It consumes the alcohol entirely, transforming it into water and carbonic acid : it also consumes the acetic acid. We must sow the M. aceti, if we do not wish to see the M. vini develop in its place, as the germs of the latter seem more widely diffused in the air. In order that the acetification may occur, the oxygen of the air is necessary. Once submerged, the M. aceti develops, but no longer produces acetic acid. It is even probable that it consumes the acetic acid already formed, reducing it to the state of water and carbonic acid. It is the same when, developing upon the surface, it has trans- formed all the alcohol. " In effect, it is not then arrested in its work ; and without changing form or mode of action, it carries the oxygen of the air to the acetic acid which it has produced, transform- ing it into carbonic acid and water. If we add some alcohol to the liquid, the phenomena change : the acid is respected, and the alcohol is transformed anew into acetic acid " (Duclaux). According to the experiments of Mayer, the maximum of aceti- fying power is obtained between 20° and 30° (68° to 86° Fah.), and this power is lost below 10° (50° Fah.) and above 35° (95° Fah.). 142 PHYSIOLOGY OF THE BACTERIA. Ammoniacal fermentation of Urine. — When urine is freely exposed to the air, we perceive at the end of a short time that it has become strongly ammoniacal. The urea is transformed into carbon- ate of ammonia by the addition of water : — CO(NH2)2 + H2O = CO2 + 2NH3. Miiller suspected that the deposit of altered urine, of which Jacquemart had already recognized the particular activity, was an organized ferment, but this was only an induction drawn from the analogy with beer yeast. Pasteur showed that this sediment is formed of a mass of spherical globules, united in chaplets, which he considers the agent of ammoniacal fermentation. These glob- ules are Micrococcus urece, Cohn, which we have already described (page 75). This bacterium lives in the interior of the liquid, and not on the surface like the Mycoderma aceti. Acidity is an obstacle to its development ; alkalin- ity, on the contrary, favors it within certain limits. Van Tieghem has even seen the fermentation con- tinue until the liquid contained thirteen per cent of carbonate of ammonia. What is the mechanism of this fermentation ? M. Musculus has shown that we may obtain from altered urine a soluble ferment upon adding to it highly-concentrated alcohol : a precipitate is formed, which may be filtered and dried. This precipitate, not at all organized, transforms urea into carbonate of ammonia. A temperature of 80° (176° Fah.) destroys it. This diastase appears, THE BACTERIA IN DIFFERENT MEDIA. 143 then, to be a secretion of the Micrococcus ureas ; and perhaps the role of the bacteria is limited, in the phenomena of fermentation, to the formation of this secretion alone. The ammoniacal transforma- tion of urine would consequently enter into the group of fermentations by the varieties of diastase. According to Arnold Hiller, if carbolic acid be added to urine, it does not become alkaline ; on the contrary, the acidity is even augmented, and that notwithstanding a considerable number of bacteria which develop in it. Has the carbolic acid killed the Micrococcus urece, leaving the field free to other organisms capable of living in an acid medium, and of producing other transforma- tions of the constituents of the urine ? In the memoir which we here cite, the author, resuscitat- ing the ancient opinion of Liebig, wishes to dem- onstrate that the decomposition of dead organic matters, and putrefaction in general, are phenom- ena purely chemical, — these decompositions being determined by the presence of organic substances, themselves undergoing transformations. We will not stop to consider these views, long since refuted : the experiments upon which they are founded are easily criticised. It is sufficient for me to say that they are in formal opposition with all the observations contained in modern works upon this question. It is especially in relation to ammoniacal fer- mentation that the question of spontaneous gen- eration has been discussed. We have already seen the results arrived at, and will not return to 144 PHYSIOLOGY OF THE BACTERIA. this subject. Let us, however, mention before closing an interesting work by MM. Cazeneuve and Livon, in which are reported some experiments which prove that urine never ferments in a healthy bladder. Lactic, Butyric, and Viscous Fermentations of Sugars. — Saccharine liquids, left to themselves, are susceptible of divers fermentations, which may occur separately or simultaneously. Those which have been best studied are three, — the lactic, the butyric, and the viscous fermentations. We will describe them successively. 1. Lactic Fermentation. — Under the probable influence of a bacterium (ferment lactique of Pas- teur) glucose and the substances susceptible of furnishing it, such as mannite, malic acid, etc., are transformed into lactic acid. From a chemical point of view, there is in this nothing more than a molecular change, lactic acid having the same composition as glucose. Taken in mass, the lactic ferment resembles beer-yeast ; its consistence is, however, a little more viscous, and its color more gray. But under the microscope, the aspect is very different, as we have seen in describing Bacterium lineola. An interesting point concerning this fermenta- tion is the action of acids upon the bacteria which produce it (presumably). As soon as the medium becomes acid, even by the lactic acid produced, the transformation is arrested. It resumes its course, if chalk or carbonate of soda is added to the liquid. THE BACTERIA IN DIFFERENT MEDIA. 145 The most suitable temperature seems to be 35° (95° Pah.). We know but little about this fermentation. " It merits, however, to be better studied. It is this which causes the spontaneous coagulation of milk : sugar of milk is transformed into lactic acid, which coagulates the caseine. We often see it occur in beef juice or in sour starch water; it must play a part in the formation of sour krout, and intervenes very certainly, and perhaps more than the alcoholic fermentation, in the preparation of bread. Finally, it very easily invades beer, which of our domestic drinks is most exposed, because of its slight acidity, to become the seat of this fer- mentation. All of these facts render it interest- ing, so much the more as it is rarely exempt from complication, and is frequently accompanied, for example, by a commencement of butyric fermenta- tion, far more disagreeable in its products" ( Du- el aux). 2. Butyric Fermentation. — This is, in fact, al- ways preceded by a lactic transformation, and it is by an ulterior modification that the lactic acid produces the butyric acid. The organism which accompanies it is a bacterium very nearly allied to Bacillus subtilis, Cohn. The reaction represented by the phenomena, from a chemical point of view, is the follow- ing : — 2CSH6O3 = C4H8O2 lactic ac. butyric ac. 146 PHYSIOLOGY OF THE BACTERIA. According to Pasteur the butyric ferment be- longs to his class of anaerobies. This fermentation resembles putrefaction in a great many particulars. Indeed some authors in- clude it under the same head. 3. Viscous fermentation. — Wines often change so that they contain a mucilaginous substance and mannite. This viscous matter has the same com- position as gum or dextrine (C6H1005) ; at the same time it disengages carbonic acid. In the fermenting liquid, we find an organism which is not yet sufficiently studied. " There are chaplets of little spherical bodies, of which the di- ameter varies sensibly, according to the kind of wine attacked by this malady (Pasteur). Pasteur has proposed the following formula : — 25(C12H22011) + 25H20 = 12(C12H20O10) + gum. 24(C6H14O6) + 12CO2 + 12H2O. ruannite. which represents the phenomena well enough as it usually occurs. There is produced in the vicinity of 51.09 of mannite and 45.5 of gum for one hun- dred parts of sugar. But sometimes the gum ex- ceeds the mannite in quantity. In this case, according to Pasteur, we can always verify in the liquid the presence of a Larger ferment of a differ- ent nature ; and the same author adds that, per- haps, in this case the increased production of gum results from the presence of this second ferment, which transforms the sugar only into gum, without THE BACTERIA IN DIFFERENT MEDIA. 147 any correlative formation of mannite. But this ferment has never been isolated. M. Monoyer has explained the variation in the proportion of gum in another manner (see his thesis for the doctorate in medicine, Strasburg, 1862). White wines are more subject than red wines to this fermentation, called graisse des vins. Accord- ing to M. Francois, the absence of tannin in the white wines is the cause of this disease, and it may be prevented by adding this substance. This remedy is even very highly appreciated in cham- pagne, according to Pasteur. What is the exact action of the tannin upon the gummy ferment? The only means of knowing is by cultivating this ferment in a state of purity and treating it with this agent. We have united together the lactic, butyric, and viscous ferments, because all three manifest them- selves in the same liquids, — wines, beer, sweetened water, etc. ; and because they have for effect the transformation of glucose. We ought to say a word here of some other inferior organisms, per- haps bacteria, observed also in the same liquids, but which have not been as well studied. Not only are they not known systematically, but we do not know precisely what is their chemical ac- tion upon the elements, of the medium which nourishes them. I shall only enumerate them. 1. Ferment of Turned Beer (Pasteur). — "These are rods or filaments, simple or articulated into chains of variable length, of about 1 ^ diameter. 148 PHYSIOLOGY OF THE BACTERIA. A high power shows them divided into a series of shorter rods, scarcely born, not yet mobile at the articulations, which are scarcely indicated." 2. Micrococcus of a beer, having a particular acidity, distinct from that of beer pique, having an acetic odor. " It consists of grains resembling little spherical points jointed by pairs or in fours square " (Pasteur), etc. § 2. — ROLE OF THE BACTERIA IN PUTREFACTION AND NITRIFICATION. While in the fermentations which we have just passed rapidly in review, we have always been able to study, at least summarily, the chemical action of the different organisms, we are now about to find ourselves in presence of phenomena far more complex. We will have to consider a great number of these vegetables at work, without its being possible to assign to each its role, or to say what is its function. The agent of the nitric fermentation has not as yet even been seen, and it is only by analogy that we class this nitrification with the true fermentations. It is not only because of the obscurity which still exists in regard to a great number of peculiar- ities of these two phenomena, that we have united them in the same study. From the point of view of the circulation upon the surface of our globe of the elements essential to the constitution of organisms, they play an analogous role, although opposite the one to the other. THE BACTERIA IN DIFFERENT MEDIA. 149 Let us consider, for example, nitrogen in plants. This element, of which the atmosphere is the res- ervoir, does not enter directly into combination, as does oxygen, with the other elements which with it are to constitute the immediate principles of the tissues. The chemical properties of nitrogen may be characterized in two words, — great resistance to entering into combination when it is free, and great facility, on the contrary, in passing from one combination to another when once it has associated itself with other elements. The circulation of nitrogen in a state of com- bination upon the surface of the globe is also an interesting question of general physics, as well as the circulation of carbonic acid, of water, and of the air. Let us seek to sketch the march of this cir- culation. Whence comes the ammonia which is found in the sea, in the clouds which come to us -from equa- torial regions, in the dust of the air ? The only known source is the fermentation of organic mat- ters out of reach of the oxygen of the air. It is to this sort of fermentation that we owe the for- mation of peat and the immense masses of com- bustible minerals which have formed during nearly all the geological periods. We see this sort of fer- mentation develop itself when we expose an or- ganic liquid to the air, but only in the inferior part of the liquid, the oxygen which is dissolved near the surface being arrested in the superficial zone, where a very different fermentation occurs. 150 PHYSIOLOGY OF THE BACTERIA. The latter is essentially oxidizing ; the material is almost completely burnt, forming water and car- bonic acid ; at the inferior part, on the contrary, a reduction is produced so energetic that hydrogen is disengaged. The metallic sulphates are there transformed into sulphites, and even crystals of sulphur are sometimes found (see the history of the Beggiatoa, page 91). We see then the source of the ammonia, which, distributed upon the soil by the winds and the rains, becomes a powerful fertilizer. Now, vegetables do not absorb nitrogen under the form of ammonia, but under the form of nitric acid. How is this transform- ation of ammonia into nitric acid effected ? The observations of Erdmann, Mensel, and T. Phipson show that in the phenomena of destructive putre- faction, nitric acid, far from being produced, is on the contrary reduced to the state of nitrous acid ; on the other hand, Th. Schloesing and A. Mlintz conclude from their experiments that in the pu- trefactions essentially oxidizing produced by Peni- cillum glaucum, Aspergillus niger, Mucor mucedo, etc., there is no formation of nitric acid. But, according to these authors, nitrification is a spe- cial phenomenon which takes place in every soil sufficiently loose to permit a free circulation of air, and of which the agent is a micro-organism. This organism has not yet been perceived, it is true; and it is evident that it would be difficult to seek and observe, because of its peculiar situation. But the action of chloroform upon nitrification tends to prove that the agent of this process is THE BACTERIA IN DIFFERENT MEDIA. 151 truly an organized ferment. Indeed, chloroform, this anaesthetic, suspends nitrification, and seems even to kill the ferment. Leaving, then, this phenomenon, but little known, we may distinguish in the agents of pu- trefaction, or more generally of fermentation, two groups of micro-organisms, — one oxidizing, the other reducing. The first are observed upon the surface of liquids undergoing putrefaction. We may distin- guish a great number of forms, — Bacterium termo, Monas crepusculum, Spirillum, etc. We ought also to include Mycoderma aceti, which, like the others, vegetates on the surface of liquids, and a great number of organisms of which we cannot speak here. The second are met, on the contrary, in the interior of liquids or of fermentable bodies; they are analogous to the butyric and lactic ferments, and perhaps to the other agents of diseases of wine and beer previously enumerated. En resume, the little beings which we have been considering have an important role : they cause the return of dead organic matter to the atmos- phere and to water. " Without them, organic matter, even exposed to the air, would not be destroyed or would be transformed with extreme slowness, in consequence of a slow combustion produced by oxygen. With them, on the contrary, its destruction takes a rapid march and becomes complete. If, then, the equilibrium is maintained between living nature 152 PHYSIOLOGY OF THE BACTERIA. and dead nature, if the air has always the same composition, if the waters are always equally fer- tilizing, it is thanks to the infinitely minute agents of fermentation and putrefaction" (Duclaux). But the role of bacteria is not limited to this. " They invade also the living organism," says Du- claux, " and bring in their attack this double char- acter of infinite smallness in the apparent means and powerful destructive energy in the results. From this source come diseases of which medicine, not long since, did not know the cause, and which she only commences to refer to their veritable origin. For those who are au courant with the first steps which she has made in this new line of research, with the fecundity of her first glimpses, with the richness of her first results, it is not doubtful that she will soon succeed in demonstrat- ing the parasitic nature of the gravest epidemic maladies " PART THIRD. TECHNOLOGY. PART FOURTH. GERMICIDES AND ANTISEPTICS. PART FIFTH. BACTERIA IN INFECTIOUS DISEASES, PART SIXTH. BACTERIA IN SURGICAL LESIONS. BY DR. G. M. STERNBERG. PLATE IX. FIG. 1. — Micrococci from bottom of culture-solution (rabbit- bouillon) inoculated with blood of septicgemic rabbit, containing the same micrococcus in active multiplication, as shown in Fig. 3. Magnified 1000 diameters by Zeiss's -^ in. horn. ol. im. objective. Methyl -violet staining. FIG. 2. — The same micrococcus cultivated in chicken-bouillon, inoculated with human saliva. X 1000. Same objective and staining. FIG. 3. — The same micrococcus as found in the blood of a rab- bit, inoculated with normal human saliva. (See p. 234.) X 1000 diameters; Zeiss's ^ in. objective. Methyl-violet staining. FIG. 4. — Micrococci from culture-solution (chicken-bouillon) inoculated with gonorrhoea! pus. X 1000 diameters ; Zeiss's y1^- in. objective. Methyl-violet staining. FIG. 5. — Micrococci from urine passed into a sterilized q-]ass vessel and allowed to stand five days, (covered with a watch-glass and bell-glass; Lister's apparatus, Fig. 5, p. 176,) believed to be identical with those shown in Fig. 3, and with Jficrococcus urece, Colin. (See description on p. 75.) X 1000 diameters; Zeiss's Jj in. objective. Aniline brown staining. FIG. 6. — Micrococci from culture-solution (malt-extract,) in- oculated with normal human saliva, probably identical with the preceding; showing multiplication in two directions. X 1000, by Zeiss's T*g in. objective. Aniline brown staining. FIG. 7. — Micrococcus urecc, from alkaline urine, showing for- mation of "chaplets," — torula-chains, — by division in one direc- tion only. X 1000, by Zeiss's Jg- in. objective. Aniline brown staining. PLATE ix. £*;:V!\. FIG. 4. IMG. 3. . 2. FIG. 5. m Jl •*•'" s £v\f»--. ••'"V'' t-"? 'J v'^^ ^5 ', • •^'V«^y\f • «»• ,4 \ * «.** «^ Ku:. 6. IMC. 7. PAET THIRD. TECHNOLOGY. to their minute size and the difficulties at- tending their study, the Bacteria received but little attention from naturalists prior to the dis- covery by Davaine of the anthrax bacillus (Com- municated to the French Academy of Sciences in 1863). Since this date, very great progress has been made in our knowledge of these minute plants ; and this progress has been due, to a consider- able extent, to the labors of physicians rather than to those of botanists, who, as a rule, have been inclined to make light of the importance attached to this and subsequent discoveries re- lating to the presence of parasitic micro-organ- isms in the blood or tissues of man and the lower animals while suffering from certain infectious dis- eases. We are greatly indebted, however, to the German botanists, Cohn and Nageli ; and to the distinguished French chemist Pasteur must be awarded the foremost place among those who have contributed to our knowledge in this direction. 156 TECHNOLOGY OF BACTERIA. As in other branches of science, progress has to a great extent been dependent upon improvements in technique. These relate especially to methods of cultivation, and to the staining, mounting and photographing of bacterial organisms. The object of the present chapter is to give as concise an account as possible of the technology as at present perfected, and as employed by the most successful modern investigators. § 1. — METHODS OF CULTIVATION. For the solution of many problems relating to the life-histories and physiological functions of the various species of Bacteria, it is essential that a " pure culture " be obtained and maintained through successive generations by the inoculation of fresh portions of a suitable culture-medium. Evidently this requires not only pure stock to commence with, but also a culture-medium free from living organisms — sterilized, — and the ex- clusion of floating atmospheric germs. Methods of OUaining Pure SlccJc. — Various meth- ods have been devised for the purpose of isolating a single species when mingled, as is commonly the case, with many others. Lister proposed to ac- complish this by diluting the material containing a number of distinct species — e. g. a drop of human saliva or of broken-down beef tea which has been freely exposed to the air — with a steril- METHODS OF CULTIVATION. 157 ized fluid until there shall be an average of less than one living germ to each drop of fluid. If now we inoculate numerous separate portions of a sterilized culture-medium with a single drop, each, of this diluted stock, it is evident that some por- tions may receive no living seed, others may have germs of two or more species, and others may chance to have one or more germs of a single species. In the latter case, the multiplication of these germs under conditions which excluded the possibility of contamination from without would give us a pure culture of this particular species. So far as the writer is aware this method has not been employed, except in a limited number of experiments made by Lister himself in order to demonstrate its feasibility. No doubt it may be successfully employed, but it would involve a great expenditure of time, and success would probably be the exception and failure the rule, owing to the difficulty of estimating the exact amount of dilution required in the first instance, and because of the element of chance, which is an essential feature of the method. The same result is accomplished more expedi- tiously by the method of Koch, the essential fea- ture of which consists in using a solid sub-stratum as the culture-medium, upon which the mixed micro-organisms are distributed. A sufficient quan- tity of gelatine (3 to 5 per cent.) is added ta a suitable culture-fluid to cause the mixture to jellify when cooled. While still warm, this gelatine cul- 158 TECHNOLOGY OF BACTERIA. ture-fluid is poured upon glass slides, to which it adheres when cool in the form of a semi-solid layer. Upon this the mixed bacteria are dis- tributed by means of a needle, the point of which is lightly drawn across the surface, after having been charged with seed by dipping it into the stock-solution a biological analysis of which is desired — e. g. broken-down urine or beef tea. The different micro-organisms are distributed by this method along the track of the needle, and the subsequent multiplication of each germ in situ, when the slide has been left for a day or two in the culture-oven, produces a little collection of the particular species to which it belongs, which may be recognized under the microscope or even by the naked eye. A pure culture is obtained by inoculating a sterilized culture-fluid with seed, transferred with due precautions, from one of these little masses formed along the track of the needle. Another method which suggests itself, and will doubtless be found useful in certain cases, depends upon the difference as to reproductive activity manifested by different species of bacteria, and upon the fact that a culture-medium, or conditions as to temperature, favorable for the development of one species may not be for another. By taking advantage of these physiological peculiarities we may succeed in excluding all but a single form, by one or more culture experiments, notwithstand- ing the fact that our stock was impure at clie METHODS OF CULTIVATION. 159 outset. It is evident that if one species multiplies more promptly and rapidly than the others which are associated with it, it will soon be present in excess in a culture-fluid inoculated with the com- mingled species, and that by using this stock to start a second culture before other forms have time to multiply, repeating the operation if neces- sary through a series of cultures, we shall at last exclude all except the single species which has taken precedence by virtue of its rapid multipli- cation. In the same way we may take advantage of conditions relating to the composition of the cul- ture medium, and to the temperature at which it is maintained after inoculation with impure stock. When the conditions are most favorable for the development of a particular species, it is evident that this will take precedence over others with which it is associated. And it may happen that conditions extremely favorable for one are entirely unsuited for other species which, accordingly, do not multiply at all. We have examples of this in the experiments which have been made upon living animals, which may be considered culture-experiments, in which the blood of the animal serves as a culture-fluid, and in which the temperature maintained is neces- sarily that of the species used in the experiment. Thus in the form of septicaemia in the mouse, which has been studied by Koch, a drop of putrid blood " containing bacteria of the most diverse 160 TECHNOLOGY OF BACTERIA. forms irregularly mixed together/' injected be- neath the skin of the animal, gives rise to an infective disease characterized by, and dependent upon, the presence of a multitude of minute ba- cilli in the blood and tissues. In this case, it is evident that the conditions are favorable for the multiplication of this species, and not for the others associated with it in the drop of putrid blood introduced into the living culture-apparatus. This experiment enables us to secure a pure cul- ture of this particular bacillus; for the smallest quantity of blood taken from the vessels of the animal, immediately after its death, contains it in abundance, and may be used to inoculate a steril- ized culture-fluid. In the same way, if we inocu- late a rabbit with a drop of human saliva, which contains a variety of bacteria, one species only multiplies freely and invades the blood of the ani- mal, producing a fatal infectious disease. This is a micrococcus of oval form and having peculiar characters. (Fig. 3, Plate IX.) By introducing a little of the blood of a rabbit, just dead as the result of such an inoculation, into a sterilized cul- ture-fluid, we obtain a pure-culture of this micro- coccus, which may be maintained indefinitely through successive generations from culture-tube to culture-tube, or from rabbit to rabbit, thus show- ing that this micrococcus is a distinct species, as it "breeds true." Having obtained pure stock by one of the methods mentioned, success in cultivating the spe- METHODS OF CULTIVATION. 161 cies contained in it will depend upon the use of a suitable culture-medium, and the maintenance of favorable conditions as to temperature and a suf- ficient supply of oxygen, if required. Natural Culture- Fluids. — The natural culture- fluids which are available for use are blood, milk, urine, and aqueous humor from the eye of one of the lower animals. All of these have been used, and all may be obtained in a pure state from the living animal by adopting proper precautions. Blood. — The observations of numerous experi- menters prove that the circulating fluid in healthy animals is free from all bacterial organisms. To obtain a supply for experimental purposes it must be drawn directly from the vessels into a sterilized receptacle. This may be accomplished by means of a glass tube drawn out at each end to form a capillary tube, hermetically sealed at each extrem- ity and thoroughly sterilized by heat. Such a tube is to be filled by exposing a superficial vein of sufficient size, and introducing one of the ca- pillary extremities within the vessel through a very small orifice made through its walls. The end of the tube is to be broken off within the vessel, after which the outer end may also be broken, to allow the contained air to escape as the tube fills with blood. This will not be necessary, however, if a partial vacuum has been formed by 11 162 TECHNOLOGY OF BACTERIA. sealing the capillary extremities in the flame of an alcohol lamp while the tube was still quite hot. Both extremities are sealed as expeditiously as possible as soon as the tube is withdrawn from the vessel. It is evident that to obtain a larger quan- tity of blood, a flask having two necks bent at a right angle and drawn out to form capillary tubes may be substituted for the simple straight glass tube. (See Fig. 1.) Fig. 1. The color of the blood, due to the presence of the red corpuscles, and the fact that these ele- ments, after a time, form a granular debris which might interfere with the recognition of minute micrococci, are objections to the use of this fluid in culture experiments. Blood-serum, how- ever, is free from these objections, and is a valua- ble culture-medium. This may be obtained from a flask, like that shown in Fig. 1, by transferring the serum, after it has separated from the clot, to small culture-flasks like those described on page 179 (Fig. 5), by the method there detailed. To accomplish this, one of the arms of the larger flask is broken off to admit the capillary extrem- ity of the smaller one. By skilful manipulation a number of these may be filled with transparent METHODS OF CULTIVATION. 163 serum with but little chance of contamination by floating atmospheric germs. Blood-serum obtained without these special pre- cautions may also be used by resorting to the method of Koch for sterilizing it subsequently to its separation from the clot. This is accomplished by introducing it into test-tubes from which at- mospheric germs are excluded by a plug of cotton, or into hermetically sealed culture-flasks, like those described on page 179, and exposing it for an hour daily to a temperature of 58° C. (136.4° Fahr.) for a period of six days. This method insures the de- struction of living germs contained in the blood- serum without coagulating the albumen, which would destroy its value as a culture-fluid. If a solid culture-medium is desired, the blood-serum is subsequently subjected to a temperature of 65° C. (149° Fahr.) for several hours. A solid, transparent, jelly is produced by this method, which is the material upon which Koch cultivated the tubercle bacillus in his experiments relating to tuberculosis. Milk. — -The experiments of Lister, Roberts and Cheyne have demonstrated that milk, as it exists in the udder of the cow, is free from the germs of fermentation or putrefaction, and may be pre- served indefinitely without undergoing change, if proper precautions are taken to introduce it into sterilized flasks without contamination by organ- isms detached from the external surface of the 164 TECHNOLOGY OF BACTERIA. body of the animal or by floating atmospheric germs. It is difficult to accomplish this, however, and in practice it will be found that inilk, although a suitable culture-fluid for various organisms, is not commonly available, owing to the difficulty of ob- taining it from its source free from contamination, and to the fact that it is a difficult fluid to sterilize. Urine. — Pasteur, Lister, the present writer, and several other experimenters have succeeded in obtaining urine, directly from the bladder, free from bacterial contamination, and which, conse- quently, did not undergo any change from being kept, although exposed freely to the air — filtered — and to a temperature suitable for inducing the different forms of fermentation which this fluid undergoes when no precautions are taken to ex- clude the micro-organisms to which these changes are due. In man, and doubtless in the lower animals also, the orifice of the urethral canal is constantly in- fested with bacteria of different species, whereas the deeper portion of the canal and the bladder are quite free from them. This is proved by microscopical examination, and by the fact that urine free from bacteria may be obtained by taking the precaution to destroy those located in the vicinity of the meatus urinarius by means of a suitable disinfectant. The writer has on several occasions repeated METHODS OF CULTIVATION. 165 with success the experiment of Lister, the essen- tial feature of which is the thorough cleansing and disinfection of the urethral canal by means of a solution of carbolic acid (5 per cent). The glans should also be washed with the same solu- tion ; after which the urine is passed into a glass flask or test-tube which has been sterilized by heat. This is at once closed with a plug of cotton. Urine has been extensively used as a culture- fluid, and is well suited for the development of many species of bacteria; and especially for the microeoccus, which has been shown by Pasteur to be the cause of the alkaline fermentation which ordinarily occurs in this fluid during warm weath- er, within a few hours after its escape from the bladder. It must be remembered, however, that decomposition of urea into carbonate of ammonia is also effected by heat, and that, consequently, the composition and reaction of this fluid is changed by boiling. For this reason its sterili- zation by heat is objectionable for certain experi- ments, and it will be necessary to obtain it from the bladder free from bacterial contamination, by the expedient above mentioned (method of Lis- ter), or by means of a sterilized catheter attached to a germ-proof receptacle, as recommended by Pasteur. Aqueous humor, obtained from the eye of one of the lower animals, recently dead, is a sterile albu- 166 TECHNOLOGY OF BACTERIA. minous fluid which has been utilized, especially by the earlier investigators, as a culture-medium. The method of operation has commonly been to place a drop of this fluid, obtained from the eye through a sterilized canula, upon a perfectly clean cover- glass, and to invert this over a shallow glass cell the margin of which has been wet with olive oil, or with a liquid cement of some kind. This serves to attach the cover and to exclude atmos- pheric organisms. The drop of fluid is inoculated by means of a needle, the point of which has been dipped into the stock-solution containing the par- ticular organism which it is proposed to culti- vate. This method is especially useful when the de- velopment of an organism is to be studied by continuous observation ; for the slide supporting a culture-cell made in this way may be placed upon the stage of the microscope, and bacteria in the drop of fluid may be observed with high powers through the thin glass cover. This method does not, however, offer as perfect security as regards the exclusion of extraneous organisms as is desira- ble, and it has generally been abandoned for the methods to be described later, in which a consid- erable quantity of fluid, enclosed in a germ-proof receptacle, is used. In this case a microscopical examination of the contained organisms requires that a small portion of the culture-fluid be with- drawn from the culture-flask, and continuous ob- servation would be impracticable. METHODS OF CULTIVATION. 167 Artificial Culture -Fluids. — The culture -fluids which have been most extensively used in in- vestigations relating to the physiology and life- histories of the various species of bacteria are infusions of animal and vegetable substances, such as beef, mutton, chicken, fish, gelatine, turnip, potato, cucumber, hay, malt, etc., etc. These in- fusions, as a rule, do not require to be very con- centrated, and they should be as transparent as possible, as the slightest opacity from suspended particles, albuminoid or inorganic, may interfere with the detection by the naked eye of changes in the fluid due to the development of bacteria, and with the recognition of these organisms upon microscopical examination. It sometimes occurs that an infusion of beef or of chicken, which has been carefully filtered and is quite transparent, becomes opalescent from the coagulation of a minute quantity of albuminoid material as the result of the operation of sterilization. I have found this opalescence difficult to remove by fil- tration. It is objectionable, but could hardly be mistaken for the opalescence, or milky opacity, which results from the breaking-down of an infu- sion of this kind, and with due care the experi- menter is not likely to be deceived, especially if he retains a portion of the sterilized fluid for comparison with that used in his culture experi- ments. Nitrogen, which is an essential element of the protoplasm of bacterial organisms, is supplied by 168 TECHNOLOGY OF BACTERIA. the albumen of animal or vegetable origin which remains in solution in the above-mentioned cul- ture-media. But this element can also be appro- priated when present in the form of ammonia, or of one of the salts of ammonia in combination with a vegetable acid. Culture -fluids may therefore be made which are suitable for the development of numerous species of bacteria, by adding to distilled water a small quantity of a salt of ammonia, together with cer- tain mineral salts, as in the formula of Mayer, given on page 113. Pasteur's solution contains ten per cent, of sugar candy and a fraction of one per cent, of ashes of yeast. (See p. 112.) Sterilization of Culture-Fluids. — Heat is the agent most available for the sterilization of culture-fluids, as chemical reagents which would accomplish the same result would also, by their presence in the fluid, prevent the development of organisms intro- duced for the purpose of cultivation. It would doubtless be possible to sterilize a fluid by means of a chemical reagent — a mineral acid for exam- ple— and subsequently to neutralize the germicide agent — e. g. by lime or magnesia. But in prac- tice it will be found that no other method is likely to give as satisfactory results as that commonly employed ; which consists in subjecting the fluid, enclosed in a germ-proof receptacle, to a tempera- ture which insures the destruction of the vitality of contained organisms. METHODS OF CULTIVATION. 169 The earlier experimenters assumed that a boiling temperature must be fatal to the minute organisms developed in organic infusions ; and this false as- sumption furnished a foundation for the belief, entertained by some of them, that bacteria might appear in such fluids by heterogenesis. The as- sumption has been proved to be false by the experiments of Pasteur, of Tyndall and of many others, and it is now known that the reproductive spores, of endogenous formation, which are devel- oped in certain species, may resist a temperature considerably above the boiling-point of water. (See p. 119.) The writer, while conducting a se- ries of experiments in the biological laboratory of Johns Hopkins University, during the summer of 1881, was greatly troubled by the fact that the laboratory was infected by the spores of a species of bacillus, which developed in little islands on the surface of his culture-fluids, even when they had been boiled for an hour or more. To destroy the spores of this bacillus, it was necessary to resort to the use of a bath of paraffine, or of concentrated salt-solution, by means of which a temperature of 105° C. was secured. This temperature, main- tained for half an hour to an hour, proved effec- tual in the destruction of these ubiquitous spores. Prolonged boiling will doubtless destroy the vi- tality of the most refractory spores ; but the exact time which is required to secure success in every case has not been determined. In practice, it will be found best to keep on the safe side, as the loss 170 TECHNOLOGY OF BACTERIA. of time and material which results from imperfect sterilization is annoying, and mistakes may arise from a false confidence in the success of the opera- tion. To avoid these, it is always best to test culture-fluids in the culture-oven for several days before using them for any experiment. The maintenance of a boiling temperature at intervals for a clay or two is more effectual than the same amount of continuous boiling. Pasteur has shown that an alkaline fluid is more difficult to sterilize than one having an acid reaction. The vitality of bacteria in active growth is destroyed by a comparatively low temperature. Thus Chau- veau has recently made the statement (C. R. Ac. des Sc., t. XCIV. p. 1694), that the anthrax bacil- lus is killed (in blood) by exposure for nine or ten minutes to a temperature of 54° (129.2° Fahr.). According to Fnsch, B. termo is killed by a tem- perature of 45° to 50° (113° to 122° Fahr.) — time of exposure not given. The writer has fixed the thermal death-point of the micrococcus of induced septicaemia in the rabbit at 60° (140° Fahr.), the time of exposure being ten minutes ; that of J//- crococcus ureae was found to be the same. The method adopted by Koch for the steriliza- tion of blood-serum for his experiments with the tu- bercle bacillus has already been mentioned (p. 165). This method depends for success upon the fact that the temperature employed, 58°, is sufficient to de- stroy growing bacteria, and that in the intervals between the daily heating for one hour the spores METHODS OF CULTIVATION. 171 have an opportunity to germinate, and are killed by the subsequent heating. The writer has not been successful in sterilizing milk by this method, and has recently lost the greater portion of a batch of tubes containing blood-serum, carefully treated according to Koch's directions, from the develop- ment of Penicillium glaucum upon the surface of the jellified serum. The spores of this fungus were evidently very abundant in the laboratory at the time the serum was introduced into these tubes, which had been well sterilized by heat and were • thoroughly protected by cotton wadding tied over the mouth of each, with the additional precaution of covering this with a piece of sheet-caoutchouc secured by a rubber band. No doubt the unusual abundance of the spores of Penicillium was due to the disturbance of the dust upon a lot of books which were taken down from an upper-shelf by my assistants, shortly before the blood-serum was decanted and introduced into the culture-tubes. According to Pasteur, the spores of Penicillium and other common mucedines are not destroyed by a temperature of 120 to 125° C (248-257° F.), in the absence of moisture. Culture Tubes and Flasks. — Glass tubes or flasks are used as germ-proof receptacles for the steril- ized culture-fluids mentioned. Ordinary test-tubes are commonly employed, and are useful for many purposes. They should be thoroughly heated in an oven, or in the flame of an alcohol lamp, just 172 TECHNOLOGY OF BACTERIA. before the fluid is introduced, to destroy all germs adhering to their inner surface. The culture-fluid may be sterilized before or after its introduction into these tubes. In the former case, the opera- tion must be performed expeditiously, in as pure an atmosphere as possible ; and the mouth of the tube is to be closed at once with a plug of cotton- wool. It is evident that this method admits of the entrance of floating atmospheric germs while the tubes are being filled, and, therefore, that a certain proportion are likely to break down. The per- centage of failures will depend upon the skill of the operator and upon the purity of the atmos- phere in wfyich the operation is performed. The liability to failure from contamination by floating germs is not, however, as great as is commonly imagined; and experience proves that contact with instruments or surfaces — e. g. the lip of the vessel from which the culture-fluid is poured — which are not perfectly pure, is a more frequent cause of the breaking-down of the culture-fluid. Sterilization of the culture-fluid after its intro- duction into the tubes, offers greater security, and the following method of manipulation is recom- mended : Test-tubes, or wide-mouthed bottles having a capacity of half an ounce or more, are washed clean, and the mouth of each is covered with several layers of cotton-wadding. This is secured in position by means of a strong linen thread, or a piece of copper wire, tied about the neck. The wide-mouthed bottles have the advan- METHODS OF CULTIVATION. 173 tage of being less fragile, and of standing without support. They are especially useful for receiving a solid culture-medium, such as gelatine solution or jellified blood-serum, as the surface exposed is greater than when test-tubes are employed. The only disadvantage attending the use of bottles is their liability to break when heated in a water- bath ; but this will not happen when Koch's meth- od of sterilization at a low temperature (140° Fahr.) is employed. The tubes, or wide-mouthed bottles, are next placed in an oven and subjected for an hour or more to as high a temperature as the cot- ton caps will bear without being scorched — about 300° Fahr. They are then cooled, and the culture- fluid is introduced, without removing the protec- tive cotton-cap, through a little funnel having a long and sharp-pointed neck, which is pushed through the layers of cotton-wadding, either di- rectly or after making a small orifice with a sharp- pointed instrument. Usually but one or two drachms of fluid will be required in each tube. This must be sterilized by heat, after its introduc- tion to the culture-tube, unless it is introduced directly from a germ-proof flask with a slender neck, such as the writer recommends for the pres- ervation of culture-fluids in bulk (Fig. 5, p. 179). In this case, the slender neck of the flask is passed through the flame of an alcohol lamp, to destroy germs which may have settled upon its outer sur- face ; and the hermetically sealed extremity is broken off with forceps which have also been 174 TECHNOLOGY OF BACTERIA. recently heated. The flask is then inverted, and the capillary neck is passed through the opening in the protective cap of a culture-tube. A suf- ficient quantity of fluid is then transferred by the application of gentle heat to the base of the in- verted flask. (See Fig. 2.) Care must be taken not to wet the protective cotton with the culture-fluid ; and immediately after this has been introduced, the ori- fice in the cotton wadding is closed by placing two more layers of the same material over those which had previously been secured to the neck of the bottle or tube. This outer protective layer may be conveniently secured in position by means of a rubber band which admits of its be- ing quickly removed for the purpose of introducing the bacteria which it is proposed to cultivate, or of extracting a drop of fluid for microscopical examination. This is ac- complished by means of a capillary tube which has been sterilized by heat just before it is used, and which is introduced through the small opening in the inner layers of the cotton cap. When tubes or bottles prepared in this way are set aside for a considerable time, or when the free admission of oxygen to the interior is not considered necessary, it is well to cover the cotton cap with a piece of thin sheet-caoutchouc, secured by means of a rub- ber band. This serves to protect the cotton cap Fig. 2. METHODS OF CULTIVATION. 175 from dust, and the contained fluid is less liable to contamination when the outer layer of cotton- wadding is removed for any purpose. It is well to carbolize the cotton-wadding used for the outer protective cap, as recommended by Lister. This is done by soaking it in a solution of one part of crys- tallized carbolic acid in one hundred parts of anhy- drous ether, after which it is allowed to dry. Lister has shown that organic infusions may be kept indefinitely, without undergoing change, in a wine-glass covered first with a watch-glass, and then with a glass shade as shown in Fig. 3. The apparatus, as arranged in the figure, is purified by being introduced into a hot oven ; and after it has cooled, the sterilized fluid is introduced from a large, double-necked stock-bottle, seen in Fig. 4. To do this, the cotton cap is removed from the nozzle of the stock-bottle, and the half of a rubber ball, having an opening in the centre, is attached to its extremity. This rubber hemisphere, which has been previously sterilized by soaking it in a strong solution of carbolic acid, serves the purpose of covering the mouth of the wine-glass when the glass cover — watch-glass — is removed. Culture-Flasks used ~by the Author. — The writer described, in a paper read at the meeting of the American Association for the Advancement of Science, in August, 1881, a method of conducting culture-experiments which he has found extremely satisfactory, and which has the advantage of as- 176 TECHNOLOGY OF BACTERIA. Fig. 3. a, wine-glass ; b, glass cover (watch-glass) ; c, bell-glass, sup- ported by a square glass plate. suring the greatest possible security from contam- ination by atmospheric germs. The culture-flasks employed contain from one to four fluid drachms. "They are made from glass-tubing of three or four tenths inch diameter, and those which the writer has used in his numerous experi- ments have all been home- made. It is easier to make new flasks than to clean old ones, and they are thrown away after being once used. Bellows, op- erated by the foot, and a flame of considerable size -gas is preferable — will be required by one who proposes to construct these little flasks for himself. After a little practice, they are rapidly made ; but as a large number are re- quired, the time and labor expended in their prepara- tion is no slight matter. . . . After blowing a bulb at the extremity of a long glass tube, of the diameter men- tioned, this is provided with a slender neck, drawn out in the flame, and the end of this is hermetically sealed. (See Fig. 5.) Thus one little flask alter another is made from the same piece of tubing, until this becomes too short for further use. METHODS OF CULTIVATION. 177 " To introduce a culture-liquid into one of these little flasks, heat the bulb slightly, break off the sealed extremity of the tube and plunge it beneath the surface of the liquid (see Fig. 6). The quantity which enters will of course depend upon the heat em- ployed, and the consequent rarefaction of the enclosed air. Ordinarily the bulb is filled to about one third of its capacity with the culture-liquid, leaving it two thirds full of air, for the use of the micro- scopic plants which are to be cultivated in it. Fig-5- " It is best not to trust to the sterilization of the culture-liquid previously to its introduction into the flasks ; for, however great the precautions taken, many failures would be sure to occur, as the result of contamination by atmospheric germs during the time occupied in the manipulations. Sterilization is therefore ef- fected by heat after the fluid has been introduced and the neck of the flask hermetically sealed in the flame of an alco- hol lamp. " This may be accomplished by boiling for an hour in a bath of paramne or of concen- trated salt solution, by which a temperature considerably above that of boiling water is secured. The writer is in the habit of 12 178 TECHNOLOGY OF BACTERIA. preparing a considerable number of these flasks at one time, and leaving them, in a suitable vessel filled with water, for twenty-four hours or longer upon the kitchen stove. Here the water-bath is kept boiling at intervals, and the contents of the flasks can scarcely fail of being subjected to a tem- perature of 212° Fahr. for eight or ten hours. When the time is less than this, failures in sterili- zation are likely to occur, and it is always best to keep on the safe side. The flasks are next placed in a culture-oven for two or three days, at a tem- perature of 35 to 38° (95 to 100° Fahr.), to $ test the success of the previous operation, — steriliza- tion. If at the end of this time the contents re- main transparent, and no film — mycodcrma — has formed upon the surface of the liquid, the flasks may be put aside for future use, and can be pre- served indefinitely. " To inoculate the liquid contained in one of these little flasks with organisms from any source, the end of the tube is first heated, to destroy germs attached to the exterior ; the extremity is then broken off with sterilized — by heat — forceps ; the bulb is very gently heated so as to force out a little air ; and the open extremity is plunged into the liquid containing the organism to be cul- tivated. The smallest quantity of this is suffi- cient, and as soon as the inoculation is effected, the end of the tube is again sealed in the flame of an alcohol lamp. A little experience will en- able the operator to inoculate one tube from an- METHODS OF CULTIVATION. 179 other; to introduce a minute quantity of blood containing organisms directly from the veins of a living animal ; to withdraw a small quantity of fluid from the flask for microscopical examination, etc., without any danger of contamination by at- mospheric germs." 1 A larger flask than those above described, hav- ing its neck drawn out in the same way, will be found the most satisfactory receptacle in which to preserve a quantity of stock solution from which to fill the smaller flasks as required. It is well not to attempt to preserve too great a quan- tity of the various organic infusions used in ex- perimental work of this kind, in a single flask ; as there is greater danger of the breaking down, and consequent loss, of the stock, when a ves- sel is frequently opened for the purpose of withdrawing a portion of its contents. It is best therefore to use a number of flasks of moderate size, rather than a single large one. There is always a saving of time and labor, when extensive experiments are contemplated, in preparing a considerable quantity of the various culture-fluids at one time, so that there may be a sufficient stock on hand in the laboratory to enable the experimenter to proceed without delay with any series of experiments he may have in view. The writer keeps constantly on hand a supply of the little flasks already described, charged with ster- 1 Extract from a paper by the Author on " The Germicide Value of certain Therapeutic Agents." The American Journal of the Medical Sciences, No. CLXX., n. s., pp. 321-343. 180 TECHNOLOGY OF BACTERIA. ilized urine, beef-tea, chicken bouillon, hay infusion, Cohn's fluid, etc., and would recommend others who may be inclined to pursue experimental investiga- tions relating to the bacteria to provide them- selves in the same way. For a reserved supply of these and other culture-fluids, flasks containing from two to four fluid ounces will be found of a convenient size. The necks of these flasks are to be drawn out in a powerful flame, so as to form a slender tube the extremity of which can be easily fused in the flame of an alcohol lamp, and which is long enough to permit of its being broken off at the end and resealed several times. The fluid is introduced into these flasks exactly as directed for the smaller ones, viz., by apply- ing heat to the body of the flask, so as to rarefy the enclosed air, and plunging the extremity of the slender neck of the flask, inverted, beneath the surface of the fluid contained in a suitable vessel. These flasks are to be hermetically sealed and sterilized exactly as was directed for the smaller ones. Each flask should have attached to it a label showing the character of its contents and the date of sterilization. Culture- Oven. — As culture experiments are com- monly conducted at a constant temperature, it is necessary to have a receptacle for the culture- tubes and flasks which can be heated artificially to any desired point, the temperature being regu- lated by a thermostat. METHODS OF CULTIVATION. 181 A rectangular copper vessel, having double walls to contain water, enclosing an air-chamber, will be found most suitable for this purpose. When the space between the double walls is filled, the air-chamber is surrounded with water on all sides, except that through which access to it is ob- tained. This side is closed by a swinging or sliding door. If the oven is of considerable size, it is well to have one or more adjustable shelves in the interior, upon which tubes and flasks may be placed, as well as upon the floor. A suitable aperture at the top admits the thermostat to the water-bath, and another aperture serves for the introduction of more water when required. A third aperture, through the centre of the upper side of the oven, leads to the air-chamber, and ad- mits of the introduction of a thermometer, the in- dex of which can be read outside while the bulb is inside of the oven. In a well-equipped laboratory several of these culture-ovens will be required, as experiments conducted at different temperatures will often be under way at the same time. The most convenient way of heating an oven of this kind is by the use of gas and of a Bunsen or other burner, which insures the complete com- bustion of the carbon. When gas is used, the thermostat described below, well known in chemi- cal laboratories, may be employed. Thermostat for Gas (Fig. 7). — The elongated glass bulb a contains a certain quantity of mer- 182 TECHNOLOGY OF BACTERIA. cury below, and air above. When the air is ex- panded by heat, the mercury rises through the __e_ tube c, which passes through the perforated cork d, and flows into the space above this cork. The tube e is connected by a piece of rubber tub- ing with a gas-jet, and the gas continues to pass through the tube f to the Bunsen burner, unless arrested by the rising of the mercury, which acts as a valve to close the lower extremity of the tube e. This tube is ad- jus table through the cork i, and it is evident that the temperature at which the gas supply is shut off will depend upon the position of its lower extrem- ity. A minute aperture in the side of the tube e permits a small quantity of gas to flow to the burner, so that the flame may not be entirely ex- tinguished when the extremity of this tube is closed by the rising of the mercury. There is dan- ger, however, when but a small amount of gas is admitted to a Bunsen burner that the flame may be extinguished by currents of air. It will there- fore be found best, in practice, to close this aper- METHODS OF CULTIVATION. 183 ture, and to have a small constant jet of gas at the side of the burner, in a position to relight the gas coming through the thermostat to the burner when the valve is opened by the falling of the mercury. The gas for this side jet does not pass through the burner or the thermostat. When the experimenter is so situated that he cannot obtain a supply of gas, the problem of regulating temperature is not quite so simple ; but the result may be accomplished by the use of a magneto-electric thermostat invented by the writer some years since. The regulating thermometer, Fig. 8, may be made as in the thermostat just described ; but, instead of a tube conveying gas, the mercury, when it rises through the tube c to the space above the cork d, meets at a cer- tain point — adjustable — the in- sulated platinum wires e and /, completing an electric circuit. A constant battery is required, — a single cup is sufficient, — and an electro-magnet, the lever of which is made, by some simple contrivance, to cut down the flame of the kero- sene or alcohol lamp used as a source of heat. This electro-magnetic regulator may also be — 05 Fig. 8. 184 TECHNOLOGY OF BACTERIA. used with gas, when great accuracy is required, by employing the valve shown in Fig. 9, which was invented by the writer for this purpose several years since. The bent tube a is con- nected with the gas supply FJg- 9- by a piece of rubber tubing. The upright arm of this tube is enclosed in a larger tube by having an outlet e, which is connected with the burner. The upper end of this larger tube is closed by means of a piece of sheet-rubber, and when this is depressed by means of the lever c, the flow of gas through the valve is arrested. The lever c has attached to it the armature <7, and is operated by an electro-magnet under the control of the regulating thermometer. To prevent the flame at the burner from being entirely extinguished every time the valve is closed, a small aperture o is made in the upright arm of the bent' tube a. § 2. THE RECOGNITION OF BACTERIA. — The breaking down of a culture-fluid, either as the re- sult of inoculation or of accidental contamination, may commonly be recognized by the naked eye. The fluid, previously transparent, may become opalescent or milky in appearance, from the pres- ence of a multitude of bacteria distributed through it ; or we may observe a pellicle upon the surface, while the fluid below remains transparent ; or, if some time has elapsed, the micro-organisms, THE RECOGNITION OF BACTERIA. 185 having exhausted the pabulum necessary for their development, may have settled to the bottom, where they form a white pulverulent precipitate, while the fluid above is transparent. In the lat- ter case, a milky appearance is produced by shak- ing the tube so as to distribute the organisms throughout the fluid. There is usually no difficulty in recognizing, by means of the microscope, the minute unicellular plants to which this change in our culture-fluid is due. But for this purpose it will often be neces- sary to use comparatively high powers, — e. g., a good one-tenth inch objective, — and to resort to the use of staining reagents. For information relating to the optical and chemical tests by which bacteria are to be distinguished from inorganic substances, and from albuminous or fatty granules, etc., the reader is referred to Part First of the present volume, which treats of their morphology, and especially to the remarks in the second chap- ter, pages 49—53. Motile bacteria are at once recognized as living organisms ; but care must be taken not to mistake movement due to currents in the fluid, or the molecular motion — brownien — which minute par- ticles undergo when suspended in a fluid, for a vital movement. This is to be distinguished by the fact that the movements are vibratory, and do not result in a change in the location or relative position of the moving particles. Bacteria which have exactly the same refractive index as the 186 TECHNOLOGY OF BACTERIA. fluid in which they are immersed, are invisible ; but if endowed with active movement, they may be detected by the disturbance they cause among motionless objects which happen to lie in their course. Thus the septic vibrio of Pasteur is so slender and transparent as to be almost invisible ; but when present in the blood of a septica3mic rab- bit, its vigorous serpentine movements are marked by a displacement of the blood globules, which it moves as a serpent moves the grass in which it is concealed. This septic vibrio I have found in the blood of rabbits, victims of my experiments in New Orleans during the summer of 1880. The use of staining reagents is indispensable for the recognition of these extremely transparent or extremely minute species. Their value has recently been demonstrated by Koch, in a most striking manner, by the discovery of a specific bacillus in the lungs and sputum of patients suf- fering with pulmonary consumption, which had escaped the observation of pathologists and mi- croscopists up to the time of his announcement of its presence and peculiar color-reaction. § 3. STAINING BACTERIA. — By far the most use- ful staining reagents are the aniline dyes, first rec- ommended by Weigert. Previously to the intro- duction of this method, hsematoxylin had been used to some extent, but did not give very satis- factory results, as " it does not stain rod-shaped bacteria at all, and only colors the spherical so STAINING BACTEBIA. 187 slightly as to prevent their certain recognition when isolated" (Koch). The aniline colors most used are the methyl- violet, aniline-brown, fuchsin, and methyl-blue. An aqueous solution of methyl-violet is perhaps the most generally useful staining fluid ; and in the violet ink sold by the stationers we have a solution ready made, which answers every pur- pose. It usually requires to be filtered. The mode of operating is as follows : The fluid con- taining the bacteria to be stained is spread in as thin a layer as possible, and allowed to dry, upon a thin glass cover. The drying may be hastened by passing the cover-glass, held in forceps, through the flame of an alcohol lamp. A drop or two of the staining-fluid is then poured upon the cover- glass, and after being left a short time is washed away by a gentle stream of water, or by agitating the cover in a glass of clean water. Usually one or two minutes is sufficient • time to ensure the staining of the bacteria attached to the cover. For immediate examination, it is now only neces- sary to place the cover on a glass slide over a little drop of distilled water. It is better, how- ever, to support the margin of the cover by means of a circle of white zinc cement, turned in the centre of the slide. This prevents the bacteria from being detached by contact with the slide. If the object is to make a permanent preparation, a drop of some preservative fluid is placed in the shallow cell formed by the circle of cement. A 188 TECHNOLOGY OF BACTERIA. saturated solution of acetate of potash, or a weak solution of carbolic acid (one per cent), or camphor water, may be used for this purpose. The surplus fluid is removed with blotting paper, and another circle of cement is turned about the margin of the cover to hermetically seal the cell. Permanent preparations may also be made by mounting in Canada balsam. In this case, the cover-glass is allowed to dry after staining, and may be treated with alcohol and oil of cloves, although this is usually unnecessary ; and too long an exposure to the action of these agents is likely to remove the color from the bacteria. To demonstrate the presence of bacteria in the tissues, the following method, devised by Weigert, is strongly recommended by Koch : — " The objects for examination are first hardened in alcohol. The sections made from these are allowed to lie for a considerable time in a pretty strong watery solution of methyl-violet. They are then treated with dilute acetic acid, the water removed by alcohol, cleared up in oil of cloves, and mounted in Canada balsam. . . . " This is of course only a general outline of the method ; for the individual tissues, and more especially the different forms of bacteria, show so great a variety of result from such treatment that it would be impos- sible to lay down rules which would be universal and which would apply to every case. For many objects fuchsin is best adapted ; for others the methyl colors are more suitable. Among these latter there exists such a difference in the staining power that the sec- tions must lie in one solution only a few minutes, in another several hours. . . . STAINING BACTERIA. 189 " The strength of the acetic acid solution is not of much consequence. The best solution is one contain- ing only a small percentage of the acid, and it is well not to allow it to act too long. The other manipula- tions, such as the removal of water, clearing up, and mounting, are exactly the same as in the preparation of other microscopic specimens. One must avoid leav- ing the sections too long in alcohol or oil of cloves ; otherwise the staining material will be washed out by these fluids." l The method above described brings to view the larger forms of bacteria which may be distributed through the tissues ; but, according to Koch, the smaller forms may not be distinguished, although deeply stained, and require for their demonstra- tion a special form of illuminating apparatus, which brings out the " color picture/' while de- tails of structure are to a great extent lost (L c. p. 27). The illuminating apparatus of Abbe, made by Zeiss of Jena, is strongly recommended by the author quoted, and will doubtless be found an important aid in difficult investigations of the nature indicated. For ordinary work, however, a good achromatic condenser will furnish the necessary illumination, and it will be found that a good one-sixth or one-tenth inch objective an- swers very well for this purpose. In order to render the number and distribution of the bacteria in an organ more evident, Koch 1 Traumatic Infective Diseases, English translation, p. 23. London, 1880. 190 TECHNOLOGY OF BACTERIA. recommends the following method. After stain- ing with an aniline color, soak the sections in a weak solution of carbonate of potash, instead of acetic acid. By this means the animal tissues, including nuclei and plasma cells, lose their color, while the bacteria alone remain stained. Staining the Tubercle- Bacillus. — The following method was first recommended by Koch : One cubic centimetre of a concentrated alcoholic solu- tion of methyl-blue is added to two hundred cubic centimetres of distilled water, and well shaken ; then add, under continuous shaking, two tenths cubic centimetres of a ten per cent solution of caustic potash. The cover-glasses upon which tuberculous sputum has been spread and dried, or thin sections of a tuberculous lung, etc., are left in this solution for twenty-four hours. If the solution is heated in a water-bath at 40° C., the staining will be effected in much less time, — half an hour to an hour. The preparation is next treated with a concentrated aqueous solution of visuvin, which should be filtered just before it is used. After one or two minutes this is washed off with distilled water. The visuvin solution discharges the blue color from the cells, nuclei and tissue elements goner- ally, giving them a brown color, while the tuber- cle-bacilli retain their blue color and are readily recognized. STAINING BACTERIA. 191 Baumgarien's Method. — In this method the spu- tum dried upon a cover-glass is moistened with a very dilute solution of potash, — one or two drops of a thirty-three per cent solution in a small watch-glass filled with distilled water. According to Baumgarten the bacilli may now be seen with a power of 400 to 500 diameters. The film of sputum is then allowed to dry, and the cover- glass is passed two or three times through the flame of an alcohol lamp, after which it is treated with an aqueous solution of one of the aniline colors. Baumgarten asserts that by this treatment the decomposition bacteria are deeply colored, while the tubercle-bacilli remain absolutely colorless. EhrlicJis Method. — This method is considered by Koch a decided improvement upon his own, and has been employed with success by numerous observers in various parts of the world, especially for the examination of sputum. This is spread upon a cover-glass in as thin a layer as possible ; and, in order to fix the albumen, the cover-glass is passed through the flame of a lamp three or four times, or kept at a temperature of 100 to 110° C. for an hour. The staining solution is pre- pared as follows : About five parts of pure aniline (" aniline oil ") are added to one hundred parts of distilled water, well shaken, and filtered through a moistened filter. A saturated alcoholic solution of fuchsin, methyl-violet, or gentian-violet, is added to this mixture, drop by drop, until pre- 192 TECHNOLOGY OF BACTERIA. cipitation commences. The cover-glass is allowed to float upon this mixture, which may be con- veniently prepared in a watch-glass, for fifteen minutes to half an hour; the side upon which the sputum has been spread is, of course, placed in contact with the staining fluid. The cover is then washed for a few seconds in a strong solu- tion of nitric acid (one part of the commercial acid to two parts of distilled water). After this it must be thoroughly washed in pure water. By this process the stain is removed from every- thing but the tubercle bacilli, which retain the color imparted to them by the first operation. The ground-substance may now be stained so as to give a strong contrast with the bacilli ; brown if the bacilli are violet, or blue if they have been stained red with fuchsin. Gills Meihod. — The following method of stain- ing the tubercle-bacillus is recommended by Dr. Gibbs, of King's College, London : — " The great advantage consists in doing away with the use of nitric acid. The stain is made as follows : Take of rosanilin hydrochloride two grammes, methyl blue one gramme ; rub them up in a glass mortar. Then dissolve aniline oil 3 c. c. in rectified spirit 15 c. c. ; add the spirit slowly to the stains until all is dissolved, then slowly add distilled water 15 c. c. ; keep in a stoppered bottle. To use the stain: The sputum having been dried on the cover-glass in the usual manner, a few drops of the stain are poured into a test-tube and STAINING BACTERIA. 193 warmed ; as soon as steam arises, pour into a watch- glass, and place the cover-glass on the stain. Allow it to remain for four or five minutes, then wash in methy- lated spirit until no more color comes away; drain thor- oughly and dry, either in the air or over a spirit-lamp. Mount in Canada balsam. The whole process, after the sputum is dried, need not take more than six or seven minutes. This process is also valuable for sections of tissue containing bacilli, as they can be doubly stained without the least trouble. I have not tried to do this against time, but have merely placed the sections in the stain and allowed them to remain for some hours, and then transferred them to methylated spirit, where they have been left as long as the color came out. In this way beautiful specimens have been made, without the shrinking which alwa}Ts occurs in the nitric acid pro- cess."— Lancet, May 5, 1883. Cheyne recommends the Weigert-Ehrlich stain- ing solution. The formula is : of a filtered watery solution of aniline one hundred parts, of a satur- ated alcoholic solution of the basic aniline dye (methyl-violet, gentian-violet, fuchsin, etc.,) eleven parts ; mix and filter. Rapid staining is obtained by warming the solution. The specimens are then decolorized by immersion in nitric acid (one part in two of water), and stained in a suitable contrast color. Very delicate sections are apt to be injured by immersion in the nitric acid. In this case, after staining them in the Weigert-Ehrlich fuchsin so- lution, they may be washed in distilled water, im- mersed in alcohol for a moment, and then placed in the following contrast stain for one or two 13 194 TECHNOLOGY OF BACTERIA. hours : distilled water 100 c. c., saturated alcoholic solution of methyl blue 20 c. c., formic acid 10 minims. According to Koch the bacillus of leprosy has the same color reaction as the tubercle-bacillus, while all other bacteria known to him differ from these in that the color imparted by one of the aniline dyes is discharged by visuvin and by nitric acid, used as above directed. The tubercle-bacilli stained by any of the meth- ods given are likely to fade after a time, especially when mounted in fluid, e. (/., glycerine or water. § 4. PHOTOGRAPHING BACTERIA. — Bacteria are prepared for photography as above directed; that is, a thin film of the material containing them is attached to a cover-glass by drying, stained, and mounted over a shallow cell containing fluid, or in balsam. For the larger forms methyl-violet is a suitable stain for this purpose; but a color less transparent for the actinic rays, such as aniline- brown or visuvin, will be required for the smaller species. The writer has given an account of the technique of photo-micrography in another work, to which the reader desiring fuller information is referred.1 It is but fair to say that satisfactory results can only be obtained by the expenditure of a consid- erable amount of time and money, as the work i Photo-Micrographs and How to make them. James R. Osgood & Co., Boston, 1883. PHOTOGRAPHING BACTERIA. 195 must be done with high powers, and the technical difficulties to be overcome are by no means incon- siderable. The illustrations in the present volume may be taken as fair samples of what may be ac- complished, and it will be found easier to criticise these than to improve upon them. Koch says, in his " Traumatic Infective Diseases " : — " In a former paper I expressed the wish that ob- servers would photograph pathogenic bacteria, in order that representations of them might be as true to nature as possible. I thus felt bound to photograph the bac- teria discovered in the animal tissues in traumatic infec- tive diseases, and I have not spared trouble in the attempt. The smallest, and in fact the most interesting, bacteria, however, can only be made visible in animal tissues by staining them, and by thus gaining the ad- vantage of color. But in this case the photographer has to deal with the same difficulties as are experienced in photographing colored objects, e. g., colored tapestry. These have, as is well known, been overcome by the use of colored collodion. This led me to use the same method for photographing stained bacteria ; and I have in fact succeeded, by the use of eosin-collodion, and by shutting off portions of the spectrum by colored glasses, in obtaining photographs of bacteria which had been stained with blue and red aniline dyes. Nevertheless, from the long exposure required and the unavoidable vibrations of the apparatus, the picture does not have sharpness of outline sufficient to enable it to be of use as a substitute for a drawing, or indeed even as evi- dence of what one sees. For the present, therefore, I must abstain from publishing photographic repre- sentations." 196 TECHNOLOGY OF BACTERIA. The difficulty of obtaining satisfactory photo- micrographs of the smallest micro-organisms is illustrated in Figures 3 and 6, Plate XI. These represent the best results which the writer has been able to attain from a large number of trials in photographing the tubercle-bacillus. In Fig. 3 there are six of these bacilli, included within an epitheloid cell, from a specimen of the sputum of a tuberculous patient. The specimen is well stained with fuchsin by Ehrlich's method ; and under the microscope the outlines of the cell, with its nucleus and the deeply-stained bacilli, are seen very dis- tinctly. But in the attempt to photograph this object it was found to be impossible to bring all of the bacilli into focus at the same time ; so that, while two bacilli are seen with tolerable distinct- ness, the others, being a little out of focus, can scarcely be distinguished. Fig. 6 represents the best result I have been able to obtain in photo- graphing a single bacillus from the same source, stained in the same way, — with fuchsin. A close inspection will show that this bacillus is formed of a chain of four oval spores. When it is re- membered that this is magnified 1,000 diameters, and has been stained and mounted secundum artem. it will not appear surprising that this minute ba- cillus escaped observation for so long a time ; and upon comparing it with the Anthrax bacillus (Fig. 1 of the same plate), we see at once a very good reason for the discovery of the latter at a much earlier date. COLLECTION OF BACTERIA. 197 § 5. COLLECTION OF ATMOSPHERIC BACTERIA. — Fully developed bacteria are rarely found in the atmosphere ; but we have ample evidence that the spores, or " germs/' of numerous species are con- stantly present, in association with the reproductive elements of plants higher in the scale, and espe- cially of the Mucorini and other microscopic fungi. Considerable attention has been given to the study of atmospheric organisms with reference to the question of their possible connection with the epidemic prevalence of certain diseases. This is not a proper place to give a summary of the results attained ; but the general statement may be made, that these have not been of a definite character, and that up to the present time no one has suc- ceeded in demonstrating, in infected atmospheres, the presence of any specific forms of bacteria which were clearly connected with the deleterious effects produced in man or the lower animals by the respiration of such atmospheres. This line of investigation, however, has by no means been ex- hausted ; and the careful and systematic study of atmospheric organisms in different localities, at different seasons, and under various circumstances as to sanitary conditions, is greatly to be desired. Any one who may be inclined to enter this field of investigation will do well to make himself familiar with what has already been done, and especially with the work of Maddox and Cunning- ham of England, and of Miquel of Paris. The last-named observer has given much time to the 198 TECHNOLOGY OF BACTERIA. enumeration of atmospheric bacteria. He finds, as might have been expected, that they are more abundant daring the summer months ; and that they are less numerous immediately after a heavy rain, which has the effect of purifying the atmos- phere, by washing out suspended particles. Rain-water will always be found fertile in germs ; and it is evident that when collected with care it represents the bacterial flora of the atmosphere at the time of its fall. We may therefore study this by means of culture-experiments, in which a variety of sterilized organic infusions are inoculated with one or more drops of rain-water which has just fallen. It is necessary to use many different culture-fluids, because various organisms require special media for their development. Again, we may expose our sterilized organic in- fusions to the air, and thus permit them to become fertilized by the deposition of air-borne germs, the development of which is subsequently studied as they germinate upon the surface, or in the interior, of these infusions. Solid culture-media are especially useful for this mode of investigation, and we may employ organic infusions to which three to five per cent of gelatine has been added, as recommended by Koch; and also a variety of cooked alimentary substances, such as moist bread, slices of boiled potato, turnip, onion, etc., various fruits (cooked or uncooked), meats of different kinds, etc. Upon the surface of these, if they are kept moist, and are placed in COLLECTION OF ATMOSPHERIC BACTERIA. 199 a culture-oven, maintained at a suitable tempera- ture, little colonies of various organisms will form from the germination of spores deposited from the atmosphere. These will soon be recognized by the naked eye, and different species may often be dis- tinguished by peculiarities as to growth, color, etc. It must be remembered that the microbes found in the atmosphere, so far as we now know, are accidentally present, and have originated else- where ; i. e., in decomposing material of organic origin from the surface of the earth. But, while we have no evidence that any known species finds the pabulum necessary for its development in the atmosphere, yet there is nothing improbable in the supposition that this may be true, and that there are species of bacteria which find in the at- mosphere all of the conditions necessary for their rapid multiplication. We know that plants much higher in the scale, which are merely attached to others for support, — epiphytes, — derive their sus- tenance directly from the atmosphere ; and it is easy to believe that, under exceptional circum- stances as to the presence of organic matter and moisture, especially in tropical climates, or during the summer months in more northern latitudes, some of these minute microscopic plants may also multiply abundantly while suspended in the atmos- phere. To judge of the relative abundance of special forms of bacteria in the atmosphere, it will be necessary to resort to direct microscopic examina- 200 TECHNOLOGY OF BACTERIA. tion of the dust deposited upon exposed surfaces, or of the suspended particles collected by means of an aeroscope. Various forms of aeroscope have been devised, the object of all being to cause a current of air to pass through a small aperture against a glass slide, the centre of which has been smeared with glycer- ine or some other viscid material, which serves to retain suspended particles. In the apparatus of Macldox, which was used by Cunningham in India, and a modification of which is employed by Miquel, a metal cone is made to face the wind by means of a weather-vane to which it is attached. A small aperture at the apex of the cone permits the con- centrated current of air to project itself against a glass slide, smeared with glycerine, which is properly supported at a short distance back of this orifice. In the apparatus used by Klebs and Tomasi-Crudeli. in their investigations in the vicin- ity of Rome, a current of air is produced by a revolving " fan-wheel " moved by clock-work. The writer, in his investigations in Havana in 1879, and in New Orleans in 1880, used a water-aspirator, by means of which a measured quantity of air was caused to flow in a given time, through a small aperture, and to impinge upon a glass slide smeared with glycerine. Any one of these methods will answer the purpose ; but the apparatus of Maddox seems to be the simplest, and has yielded very satisfactory results. Instead of collecting the suspended organisms ATTENUATION OF VIRUS. 201 by means of a drop of glycerine attached to a glass slide, Pasteur has proposed to collect them by passing a current of air through a glass tube containing a loosely-packed filter of gun-cotton. This is subsequently dissolved in ether, and, upon evaporation of the ether, the participate atmos- pheric impurities are found in the film of collodion remaining. Examination of Water. — The bacterial flora of water from any source may be studied by the method already referred to in speaking of rain- water ; viz., by using a small quantity to inoculate a variety of sterilized organic infusions^ and ob- serving the development of the various micro- organisms which make their appearance as the result of this procedure. Dr. Angus Smith of Manchester has recently given a favorable account of results obtained by the gelatine method proposed by Koch. Pure fish- gelatine is added to the water to be tested, in sufficient quantity to form a gelatinous mass. If the water is pure, this remains for a long time un- altered ; but. if impure from the presence of living organisms, the gelatine becomes liquefied in the vicinity of these, and little bubbles are formed, at the bottom of which the bacteria will be found. § 6. ATTENUATION" OF VIRUS. — Various methods of producing physiological varieties of pathogenic bacteria, to be used in protective inoculations, have 202 TECHNOLOGY OF BACTERIA. been proposed since Pasteur first announced (1880) that the microbe of fowl-cholera could be modified, by special treatment, in such a manner that it no longer produced a fatal form of the disease ; and that fowls inoculated with this " attenuated virus " were subsequently protected against the disease, resisting inoculation with the most potent virus. Method of Pasteur. — Pasteur found that the poison of fowl-cholera was most virulent when ob- tained from fowls which had died from a chronic form of the disease, and that this virus could be cultivated in chicken-bouillon for many successive generations without any diminution of its potency, if the interval between two successive inoculations was not greater than two months. But when a greater interval than this was allowed to elapse, the disease produced by inoculation was of a less serious character, and fewer deaths occurred. This diminution of virulence became more marked in proportion to the length of time during which a culture-solution containing the microbe remained exposed to the action of the atmosphere, and at last all virulence was lost, as a result of the death of the parasite. That this result is due to contact with the oxygen of the air is shown by the fact, that virus enclosed in sealed tubes does not undergo this modification, but retains its full virulence for many months. According to Pasteur, the various degrees of modification of virulence produced by prolonged exposure to oxygen are preserved by ATTENUATION OF VIRUS. 203 the cultivation, at short intervals, of the different grades of " attenuated virus." Subsequent experiments with the virus of an- thrax (charbon) gave similar results ; and, under the direction of Pasteur, extensive protective inocula- tions have been practised in France with attenu- ated virus prepared by this method. The time of exposure to oxygen is less for the anthrax bacillus than is required in the case of the micrococcus of fowl-cholera ; and it is necessary to cultivate the bacillus in such a way as to pre- vent the development of spores, as these retain their virulence unchanged for many years. This is accomplished by cultivating the bacillus at a temperature of 42° to 43° C., at which point no spores are developed, the organism multiplying by fission only. Contact with the atmosphere for a month destroys entirely the vitality of the bacillus in such a culture, and in eight days it loses its deadly properties, — the temperature being main- tained at the point mentioned. During this time the virus passes through successive degrees of at- tenuation. It is possible to restore the mitigated virus to its full activity by inoculating a guinea- pig one day old, which is killed by the operation, and using the blood of this animal to inoculate a second ; and so on. After repeating this operation several times, the poison is said by Pasteur to re- gain its full vigor, and to be fatal to a sheep. In the same way the attenuated virus of fowl-cholera may be restored to full vigor by inoculating a 204 TECHNOLOGY OF BACTERIA. small bird, — sparrow or canary, — to which it is fatal. After several successive inoculations from bird to bird, the virus resumes its original activity. Method of Toussaint. — The effect produced upon pathogenic organisms by prolonged exposure to oxygen, Toussaint proposes to produce more ex- peditiously, by subjecting them for a short time to a temperature a little less than is required for the complete destruction of vitality. According to Chauveau, this is best accomplished, in the case of Bacillus anthracis, by exposure for eighteen minutes to a temperature of 50° C. Ex- posure to this temperature for twenty minutes is said to kill the bacillus ; while " heating for eighteen minutes produces an excellent attenuated virus for vaccination." A first vaccination with feeble virus (heated to 50° for fifteen minutes), and a second inoculation, at the end of fifteen days, with a strong virus (blood heated to 50° for nine or ten minutes), preserves sheep from the effects of subsequent inoculations with virus of full strength. The heating must be in small tubes, not more than 1 mm. in diameter ; and at the end of the time fixed these must be quickly withdrawn from the hot bath and plunged into cold water. The blood of a guinea-pig which has just died from anthrax, at the end of thirty-six to forty-eight hours from the time of inoculation, is said to be a good active virus upon which to operate by this ATTENUATION OF VIRUS. 205 method. The attenuated virus, when used to in- oculate a culture-fluid, develops more or less rapidly, according to the degree of attenuation. Bacilli heated for the longest time, and those sub- jected to the highest temperature, are the longest in showing signs of development. Method of Chauveau. — Chauveau has attempted to test experimentally the question whether sus- ceptible animals might not resist infection by a small number of active bacilli, and acquire im- munity as the result of such inoculation. His results were favorable to the view that this is true as regards anthrax, at least ; and Salmon has since adduced satisfactory evidence that it also applies to fowl-cholera. The method adopted by Chau- veau consisted in diluting infected blood from the guinea-pig until a cubic centimetre of the mixture contains, as nearly as can be computed, the num- ber of bacilli desired. A given quantity of this fluid was injected into the jugular vein of a sheep. Sheep of native French breeds were invariably killed when the number of bacilli introduced into the circulation was about one thousand. In an experiment in which two hundred and fifty bacilli were injected into each of five sheep, all with- stood the dose, and four showed immunity when reinoculated at the end of six weeks. Immunity against symptomatic anthrax was also procured by the same procedure. Salmon, who has tested this method in fowl-cholera, has arrived at the follow- ing conclusions : — 206 TECHNOLOGY OF BACTERIA. "First. — A single disease-germ cannot produce this extremely virulent disease ; it cannot even multiply sufficiently to produce the local irritation at the point of inoculation. When a quantity of virus was intro- duced into the tissues, which should have contained at least twelve germs, there was no effect, either general or local ; but by increasing this one third, with the same birds, the local irritation appeared. kt Second. — It is apparent that the local resistance to the germs fails, while the constitutional resistance may still be perfect, and that in this case there may be a local multiplication of the organisms for two or three weeks without any disturbance of the general health. " Third. — That this local multiplication of the virus is sufficient to grant a very complete immunity from the effects of such virus in the future." 1 Method In Intravenous Injection. — In sympto- matic anthrax, it has been found by Aiioing, Cornevin, and Thomas, that intravenous injection of the virus produces in the calf, the sheep, and the goat only a slight indisposition, lasting for two or three days ; and that subsequently the tumors characteristic of this disease are not developed as the result of inoculation in the muscles with the bacterium to which the disease is ascribed. Attenuation of Vims tyj Chemical Reagents. — The attenuation of virulence which results from ex- posure to oxygen (method of Pasteur), or to an elevated temperature (method of Toussaint), seems to depend upon diminished reproductive activity i The Med. Record, April 7, 1883, p. 371. ATTENUATION OF VIRUS. 207 of the pathogenic organism. Evidently the tis- sues of a susceptible animal are able to resist the invasion of a limited number of active germs (di- lution of virus), and of a still greater number of those which are less active as a result of the treatment referred to. The writer has obtained evidence, in the course of his experiments relating to the comparative value of disinfectants, which goes to show that certain chemical reagents, also, may modify the virulence of pathogenic bacteria in a similar man- ner. In these experiments, the blood of a rabbit recently dead from induced septicaemia was the virulent fluid used as a test. The pathogenic or- ganism in this case is a micrococcus, which is found in normal human saliva. In the published report of these experiments the following state- ment is made : — " The most important source of error, however, and one which must be kept in view in future experiments, is the fact that a protective influence has been shown to result from the injection of virus, the virulence of which has been modified, without being entirely destroyed, by the agent used as a disinfectant." l Sodium hyposulphite and alcohol were the chem- ical reagents which produced the result noted in these experiments ; but it seems probable that a variety of antiseptic substances will be found to be equally effective, when used in the proper pro- 1 Studies from the Biological Laboratory, Johns Hopkins University, Vol. II. No. 2, p. 205. 208 TECHNOLOGY OF BACTERIA. portion. Subsequent experiments have shown that neither of these agents is capable of destroying the vitality of the septic micrococcus in the pro- portion used (one per cent of sodium hyposulphite and one part of ninety-five per cent alcohol to three parts of virus), and that both have a re- straining influence upon the development of this organism in culture-fluids. PART FOURTH. GERMICIDES AND ANTISEPTICS. A KNOWLEDGE of the vital resistance of the various species of bacteria to the action of differ- ent chemical reagents is important from several points of view. First, such information has an important bearing upon elementary biological problems, which are best studied in these simple unicellular plants; second, practical sanitation, and the preservation of various food-products, depend to a considerable extent upon the proper use of germicides and antiseptics; and, iliird, modern therapeutics has been largely influenced by the indications which this knowledge seems to furnish for the treatment of infectious diseases and surgi- cal injuries. By a germicide agent we mean one which has the power to destroy the vitality of the various species of bacteria known to us, including those disease-germs which have been demonstrated, such as the anthrax bacillus, the bacterium of symptomatic anthrax, the micrococcus of fowl- cholera, that of septicaemia in the rabbit, etc. 14 210 GERMICIDES AND ANTISEPTICS. Germicides are also antiseptics, as the bacteria of putrefaction are killed by them as well as those mentioned. They may also arrest putrefactive decomposition in quantities less than are required to completely destroy putrefactive organisms. But an antiseptic is not necessarily a germicide ; for experiment proves that certain substances ar- rest putrefaction which have not the power to kill the bacteria to which this is due. This they do by arresting the vital activity — multiplication — of the germs of putrefaction, or by so changing the nutritive pabulum required for the develop- ment of these germs that they are unable to appropriate it to their use. If it were proven that the infectious character of every kind of infective material depended upon the presence of a specific living germ, as has been shown to be true in the case of certain kinds of infective material, germicide and disinfectant would be synonymous terms. Although this has not been proved, it is a significant fact that all of the disinfectants of established value have been shown by laboratory experiments to be potent germi- cides. The antiseptic value of a substance is readily determined by a series of experiments in which it is added in various proportions to putrescible or- ganic substances, and observing if, under favorable conditions as to temperature and moisture, putre- faction is arrested or prevented. Some observers have made arrest of motion in GERMICIDES AND ANTISEPTICS. 211 the motile bacteria a test of germicide power. But it is evident that this is unreliable, and the only safe test is failure to multiply, under favora- ble conditions, in a suitable culture-fluid. This test requires care in its application, as contamina- tion of the culture-fluid by other organisms than those which have been subjected to the action of the germicide agent would give a misleading result. The method adopted by the writer in a series of experiments, the results of which are published in the "American Journal of the Medical Sciences," April, 1883, is very satisfactory and reliable. This consists in the use of the little culture-flasks, con- taining a sterilized organic infusion, prepared as directed on p. 179 of the present volume. The bacteria which serve as a test are subjected to the action of the germicide in a small glass tube, previously sterilized by heat; and, after a given time, which in the experiments referred to was two hours, the fluid in the culture-flask is in- oculated with a minute drop of fluid from the tube containing the test-organisms. The culture-flask is then placed in the oven, at a temperature of 98°- 100° Fahr. At the end of twenty -four to forty-eight hours, inspection of the little flask will show in a very definite manner whether the ger- micide has been effectual or not: for the fluid will remain unchanged and transparent if the test- organisms were killed by the germicide agent ; or, in case of failure, will have broken down, and will 212 GERMICIDES AND ANTISEPTICS. present an opalescent or milky appearance, from the abundant development which has taken place as the result of inoculation. When a pathogenic organism is used to test the germicide power of chemical substances, we may inoculate living animals instead of sterilized culture-fluids. In this case, failure to produce the characteristic symptoms of the disease is, of course, to be taken as evidence that the vitality of the pathogenic germs was destroyed before inocula- tion. The most available organisms for such ex- periments, in the present state of science, are the bacillus of anthrax, the micrococcus of fowl-chol- era, the bacterium of symptomatic anthrax, and the micrococcus of induced septicsemia in the rabbit. In a series of experiments made by the writer in 1881, the last-named organism, as found in the blood of a rabbit recently dead, served as the test. The results were on the whole quite satisfactory and definite ; but there are certain sources of error connected with this method which should be borne in mind. First. The test-organism may be modi- fied as regards reproductive activity without being killed ; and, in this case, a modified form of dis- ease may result from the inoculation, of so mild a character as to escape observation. Second. An animal which has suffered this modified form of disease, enjoys protection, more or less perfect, from future attacks, and if used for a subsequent experiment may, by its immunity from the effects GERMICIDES AND ANTISEPTICS. 213 of the pathogenic test-organism, give rise to the mistaken assumption that this had been destroyed by the action of the germicide agent to which it had been subjected. Vaccine virus has also been used by the writer, and by other experimenters, to test the compara- tive value of disinfectants. The method consists in dividing a certain quantity of virus from the same source into two parts, and subjecting one portion to the action of the agent to be tested, while the other is reserved to prove the reliability of the material used. A negative result from vac- cination with the disinfected virus, and a positive result from that not disinfected, is evidence of the power of the disinfectant used to destroy the in- fective virulence of the material. The experiment must of course be made upon un vaccinated chil- dren, and it is best to make it in duplicate, two punctures being made upon one arm with the disinfected virus, and two in the other with that not disinfected. A complete resume of the experiments which have been made to determine the value of anti- septics and disinfectants would require more space than can be given to this subject in the present volume. Nor can the results obtained by different methods be brought together in tabular form ; for discrepancies exist, due to various circumstances, and an extended discussion wrould be required to reconcile these, or to determine which wrere en- titled to the greatest consideration. These dis- 214 GERMICIDES AND ANTISEPTICS. crepancies arise from the following circumstances : (a) The different bacteria which have been used as test-organisms differ within certain limits as regards vital resistance to the action of germicide agents. A like difference may occur in a particu- lar species (b) as the result of the presence or absence of reproductive spores ; (c) because of dif- ferent conditions relating to the physical character of the material containing the germs; e.g., solid or fluid, coagulated masses, etc; (d) from a differ- ence in the reaction of the media in which they are contained ; (e) from a difference in the time of exposure to the action of the reagent. The list which follows is arranged, for con- venience, in alphabetical order. The writer has given his own results the precedence ; and, as his experiments were made with special care by a method which offers the greatest possible security against error, he believes that they will be found, in the main, to be trustworthy. The letter S, enclosed in brackets, will be used to designate these ; while results obtained from other sources will be followed by the name of the experimenter who has reported them. In the author's experiments, unless otherwise stated in the text, the time of exposure to the action of the germicide agent was two hours. The septic micrococcus, frequently referred to below as one of the test-organisms employed, is from the blood of a rabbit recently dead, as the result of inoculation with human saliva ; and, when GERMICIDES AND ANTISEPTICS. 215 " septicnemic blood" is spoken of, the blood of a rabbit which has fallen a victim to this form of septicaemia is meant. (Consult bibliography for titles of papers by the writer relating to this form of induced septicaemia in the rabbit.) Acetic Acid. — This has the lowest preventive power in Group II. — the Organic Acids (Dougall). Alcohol ranks low as a germicide, but is not with- out value as an antiseptic. Exposure to ninety- five per cent alcohol for forty-eight hours did not kill the bacteria in broken-down beef- tea (old stock). The septic micrococcus was destroyed by two hours' exposure to a twenty-four per cent solution. The micrococcus of gonorrhoeal pus required a forty per cent solution (S). " Pare or camphorated alcohol is largely used by surgeons in France to wash their instruments, but is evidently capable of giving only an illusory safety against morbid germs. . . . When saturated with camphor, alcohol does not destroy the virus of symptomatic anthrax" (Arloing, Cornevin, and Thomas). In the proportion of 1: 1.5, it destroys the bacteria which cause the acid fermentation of milk (Molke). 1: 1.18 destroys the bacteria of broken-down beef-tea, and 1 : 20 prevents the de- velopment of these bacteria in sterilized beef- infusion (de la Croix). The micrococcus of pus multiplies freely in a culture-fluid containing five per cent of alcohol, but fails to multiply in a so- lution containing ten per cent. Exposure for half an hour to alcohol in the proportion of twelve per 216 GERMICIDES AND ANTISEPTICS. cent did not destroy the virulence of septic blood, which was injected into a rabbit with a fatal result. Twice this amount, however, proved effectual (S). Aluminium Acetate. — The development of bac- teria in pease-infusion is prevented by 1: 5,250 (Kiihn). The development of bacteria in un- boiled beef-infusion was prevented by 1: 6,310; and the bacteria of broken-down beef-tea were destroyed by 1: 478, while 1: 584 failed (de la Croix). Aluminium Chloride. — " Group III. — Salts of the Alkaline Earths. Here chloride of aluminium is highest. . . . Were it not for the extremely high preventive point (1: 2,000) of this salt in the hay column, this group would occupy a compara- tively subordinate position" (Dougall). Ammonia does not destroy the virus of sympto- matic anthrax (Arloing, Cornevin, and Thomas) ; or the spores of the anthrax bacillus (Koch). Aromatic Products of Decomposition. — Bauman first showed that phenol is developed in albuminous fluids during the process of putrefaction; and Sal- kowski found, in 1875, that old putrid fluids have antiseptic properties. Wernich has studied this subject, and finds that the aromatic products of decomposition, — skatol, phenyl, propionic acid, in- dol, kresol, phenyl acetic acid, and phenol, — arrest putrefaction, when present in organic infusions in small quantities, in the order named. Arsenious Acid. — One per cent destroys spores of bacilli in ten days (Koch). GERMICIDES AND ANTISEPTICS. 217 Benzoic Acid. — One part in 2,000 retards the development of spores (Koch). One part in 1,439 prevents development of bacteria in unboiled meat- infusion ; 1 : 2,010 does not. The bacteria of broken- down beef- tea are destroyed by 1 : 77, while 1 : 121 failed (de la Croix). In Group II. — the Organic Acids — benzoic has the highest prevent- ive power (Dougall.) Boric Acid in saturated aqueous solution (four per cent) failed to destroy the three test-organisms employed in the writer's experiments. But it pre- vented the development of the M. of pus in the proportion of 1 : 200 ; of the M. of septicaemia in 1 : 400, and of B. tenno in 1 to 800. This differ- ence, as regards ability to multiply in the presence of boric acid, accounts for the fact that micrococci have been observed to be present in the pus of wounds treated antiseptically with this substance, although no evidence of putrefaction could be dis- covered. A two per cent solution destroyed the virulence of septicsernic blood ; but, in view of the fact that twice this amount did not kill the micro- coccus to which this virulence is due, it is evident that the result obtained in inoculation experiments upon rabbits was due to the restraining — anti- septic— power of the reagent, and can not be taken as evidence of germicide power (S). The activity of fresh virus of symptomatic anthrax was destroyed by boric acid, one in five (twenty per cent) the time of exposure being forty-eight hours (Arloing, Cornevin, and Thomas). One part 218 GERMICIDES AND ANTISEPTICS. in 133 prevented the development of bacteria in tobacco-infusion, while 1 : 200 failed (Bucholtz). One part in 58 prevented the development of bac- teria in a vegetable infusion (peas), while 1 : 81 failed ; 1 : 101 failed to preserve a solution of egg- albumen (Klihn). A five per cent solution was found by Koch, to be inert, the test being the anthrax bacillus. Bromine. — The spores of bacilli are killed by a two per cent aqueous solution of bromine. In the form of vapor this agent is superior, as regards rapidity of action, to chlorine and iodine (Koch). Bromine vapor is the most active agent for the destruction of the virus of symptomatic anthrax (Arloing, Cornevm, and Thomas). It destroys the ferment of sour milk (Bacterium lactis] in the pro- portion of 1 : 348 (Molke). The bacteria of broken- down beef-tea are destroyed by 1 : 336 ; and the development of bacteria in unboiled meat-infusion is prevented by 1 : 5597 (de la Croix). Camphor does not destroy the infective proper- ties of vaccine except when it is exposed for at least a week in an air-chamber saturated with the volatile oil (Braid wood and Vacher). Alcohol sat- urated with camphor has no action upon the fresh virus of symptomatic anthrax (Arloing, Cornevm, and Thomas). One part to 2,500 retards the de- velopment of anthrax spores (Koch). Carbonic Acid. — Of five experimental vaccina- tions with lymph subjected to this gas, three succeeded (Braidwood and Vacher). GERMICIDES AND ANTISEPTICS. 219 Carbonic Oxide. — Vaccine lymph may endure at least twenty-four hours' exposure to carbonic ox- ide without losing its specific properties (Braid- wood and Yacher). This gas has no effect upon bacteria, which readily develop in it (Hamlet). Carbolic Acid, in the proportion of one to two hundred, destroys B. termo and the septic micro- coccus in active growth, while 1 : 25 failed to de- stroy the bacteria in broken-down beef-tea (old stock) ; the micrococcus of pus was destroyed by 1 : 225. The development of all of these organ- isms was prevented by the presence in a culture- fluid of 0.2 per cent = 1 : '500 (S). The micro- coccus of swine plague multiplies abundantly in urine containing 1 per cent of carbolic acid, while the micrococcus of fowl-cholera is destroyed by six hours' exposure to a 1 per cent solution (Sal- mon). A 2 per cent solution destroys the bacte- rium of symptomatic anthrax (dried virus), the time of exposure being forty-eight hours (Ar- loing, Cornevin, and Thomas). The multiplica- tion of bacteria in urine is not prevented by 1 : 100 (Haberkorn). In egg-albumen develop- ment of bacteria is prevented by 1 : 200 (Ku'hn). One part to 502 prevents the development of bacteria in unboiled meat-infusion; but the bac- teria in broken-down beef-tea are not destroyed by a 10 per cent solution (de la Croix). A 5 per cent solution required two days to arrest the developing power of the spores of Bacillus anthracis, while a 1 per cent solution destroyed the bacilli 220 GERMICIDES AND ANTISEPTICS. themselves in two minutes. A solution of 1 : 850 prevented the multiplication of these bacilli in a suitable culture-medium. Carbolic acid in solu- tion, in oil or in alcohol, is without effect upon the spores of B. anthracis, which germinated after being immersed 110 days and 70 days, respec- tively, in a 5 per cent solution in oil and in alco- hol (Koch). The same author found that car- bolic acid vapor, at 75° C., for two hours, failed to destroy anthrax spores. Chemical combina- tions with other substances were less efficacious than the pure acid. A 5 per cent solution of zinc sulpho-carbolate destroyed anthrax spores in five days ; a 5 per cent solution of sodium phenate, in ten days, merely reduced their power of develop- ment, while sodium sulpho-carbolate failed to do this within the same time. Chloral Hydrate failed to kill the micrococcus of pus in the proportion of 10 per cent, but was successful in the proportion of 20 per cent (S). Chloroform. — A comparatively brief exposure to chloroform vapor entirely sterilizes vaccine lymph (Braid wood and Yacher). Chloroform has no effect upon the fresh virus of symptomatic anthrax (Arloing, Cornevin, and Thomas). Chloroform is inert as regards the destruction of the spores of the anthrax bacillus (Koch). The development of bacteria in unboiled beef-infusion is prevented by 1 : 103; but 1 : 1.22 failed to destroy the bac- teria of broken-down beef-tea (de la Croix). Chlorine.— Exp. No. 37, Jan. 27, 1880.— Four GERMICIDES AND ANTISEPTICS. 221 children were vaccinated with virus from ivory points which had been exposed for six hours to an atmosphere containing one half per cent of chlo- rine (produced by the action of hydrochloric acid on the peroxide of manganese, and collected over warm water) ; also with four points, from the same lot, not disinfected. Result : Vaccination was un- successful in every case with the disinfected points, and successful with those not disinfected (S). Chlorine destroys the fresh virus of symptomatic anthrax, but is powerless against that which has been dried (Arloing, Cornevin, and Thomas). Chlorine is classed with bromine, iodine, and corrosive sublimate, as one of the most relia- ble agents for destroying the spores of anthrax (Koch). Development of bacteria in unboiled beef-infusion is prevented by the presence of one part in 15,606, and the bacteria of broken-down beef-tea are destroyed by 1 : 1,061 (de la Croix). Chromic Acid, in the proportion of 1 : 1000, de- stroys the virulence of septicaemic blood (S). The development of anthrax spores is prevented by 1 : 5000 ; but chromic acid and its salts are ineffi- cient for the destruction of these spores (Koch). Chromic acid was found to have a preventive power surpassing all others, its average being 1 : 2,200, while that of carbolic acid is only 1 : 226 (Dougall). Citric Acid., in the proportion of 12 per cent, proved fatal to the micrococcus of pus, while 10 per cent failed (S). 222 GERMICIDES AND ANTISEPTICS. Creosote, in the proportion of 1 : 200, is fatal to the micrococcus of pus (S). Cupric Sulphate destroys the virulence of septi- csemic blood in the proportion of 1 : 400 (S). The activity of dried virus of symptomatic anthrax is destroyed by a 20 per cent solution — time of exposure forty-eight hours (Arloing, Cornevin, and Thomas). The metallic salts, from their showing the highest average preventive power, form Group I. Sulphate of copper here has not only the highest individual average, but its three pre- ventive points, in the three solutions, are very much higher than those of any other substance in the group (Dougall). Ether does not destroy the spores of bacilli after thirty days' exposure (Koch). A brief exposure to the vapor of ether destroys the infective power of vaccine lymph (Braidwood and Vacher). Eucalyptol retards the development of the spores of bacilli in the proportion of 1 : 2,500 (Koch). In the proportion of 1 : 205, the development of bac- teria in unboiled meat-infusion is prevented. The bacteria in broken-down beef-tea are not destroyed by 1 : 14 (de la Croix). Ferric Sulphate. — A saturated solution of this salt did not kill any of the test-organisms, and the use of this agent as a disinfectant would evidently be a serious error. It has, however, a decided value as an antiseptic, having prevented the de- velopment of all of the test-organisms in the pro- portion of 1 : 200. Although not fatal to the GERMICIDES AND ANTISEPTICS. 223 septic micrococcus in the proportion of 16 per cent, it prevents the development of septicaemia in the rabbit, after inoculation with septic blood to which it has been added, in the proportion of 1 : 400 (S). Exposure to a 20 per cent solution for forty-eight hours did not destroy the virus of symptomatic anthrax (Arloing, Cornevin, and Thomas). Fern Chloridi Ttnct. — A 4 per cent solution was fatal to the two species of Micrococcus , but failed to kill B. termo. The micrococci were not destroyed by a 2 per cent solution (S). Glycerine , in the proportion of 12.5 per cent, destroyed the virulence of septicaemic blood, but failed at 10 per cent (S). Glycerine has no action upon the fresh virus of symptomatic anthrax (Arloing, Cornevin, and Thomas) ; and is inert as regards the spores of bacilli (Koch). Heat. — The thermal death-point of the micro- coccus of septicaemia (induced septicaemia in the rabbit) is 140° Fahr. (60° C.), the time of ex- posure being ten minutes ; that of the micrococ- cus of gonorrhoeal pus (believed to be identical with M. weae, Cohn), is the same (S). The micrococcus of fowl-cholera is destroyed by expo- sure for fifteen minutes to a temperature of 132° Fahr. (Salmon). Nine or ten minutes' exposure to a temperature of 54° C. is sufficient to com- pletely kill the bacilli in anthrax blood (Chau- veau). Colin has assigned 55° C. as the highest point at which bacteria studied by him have lived and developed. * Van Tieghem says that this tem- 224 GERMICIDES AND ANTISEPTICS. perature is fatal to most of these organisms ; but he has studied a bacillus which is able to multiply and form spores in a culture-fluid at a tempera- ture as high as 74° C., but which ceased to mul- tiply at 77°. Miquel had previously reported the existence, in the water of the Seine, of an im- mobile filamentous Bacillus, whch supports a tem- perature of 70° C., and which he has cultivated at this temperature in a neutral meat-infusion. This Bacillus was killed by a temperature of 71° to 72° C. The spores of B. subtilis resist for several hours a temperature of 100° C. (212° Fahr). The time required to kill these spores varies according to the nature of the liquid. In yeast-water, and in hay- infusion, they can resist a boiling temperature for five hours ; while in distilled water they are killed after two or three hours. A temperature of 115° C. kills them very quickly (Chamberland). Desiccated septic blood does not lose its virulence at the end of forty days ; or by being heated to 100° for from three to twenty-four hours, and the contained bacteria are capable of multiplication after such exposure (Lebedeff). Hydrochloric Acid, in the proportion of 1 : 200, destroys the virulence of septicoemic blood (S). Hydrochloric acid gas destroys the contagion of vaccine (Braidwood and Vacher). A 2 per cent solution of muriatic acid kills the spores of the anthrax bacillus in ten days, while the develop- ment of these spores is prevented by 1 : 1,700 (Koch). GERMICIDES AND ANTISEPTICS. 225 Hydrogen. — Bacteria may develop in an atmos- phere of hydrogen (Hamlet). Iodine (in aqueous solution with potassium iodide) destroys the septic micrococcus in the proportion of 1:1,000; the micrococcus of pus and B. termo in 1 : 500. It prevents the develop- ment of these organisms when present in a culture- solution in the proportion of 1 : 4,000 (S). The development of bacteria in tobacco-infusion is prevented by 1 : 5,714 (Bucholz); in boiled beef- infusion, 1 : 2,010; in unboiled, 1:10,020 (de la Croix). One part in 1,000 destroys the bacteria which produce the acid fermentation of milk (Molke) ; and 1 : 410 the bacteria of broken-down beef-tea (de la Croix). Mercuric Bichloride. — All experimenters agree in placing this in the front rank as a germicide and antiseptic agent. One part in 40,000 prevents the development of the septic micrococcus, and but little less is required in the case of the micro- coccus of gonorrhoeal pus and of B. termo. To destroy the vitality of bacteria in broken-down beef-tea (old stock) required 1 : 10,000, while the above-mentioned micrococci were killed by 1 : 20,- 000 (S). The activity of the virus of sympto- matic anthrax (dried virus) is destroyed by 1: 5,000 (Arloing, Cornevin, and Thomas). The bacteria of broken-down beef-infusion are destroyed by 1 : 6,500, and their development in beef-tea prevented by 1 : 10,250 (de la Croix). One part in 20,000 prevents the development of bacteria in sterilized 15 226 GERMICIDES : AND ANTISEPTICS. tobacco-infusion (Bucholz). One part in 1,000 destroys the spores of Bacillus anthmcis in ten minutes (Koch). Nitric Acid in the proportion of 1 : 400 destroys the virulence of septicsemic blood — time of con- tact, half an hour (S). Nitrous Acid. — Exp. No. 36, Jan. 22, 1880.- Three children were vaccinated with ivory points which had been exposed for six hours to an at- mosphere containing one per cent of nitrous acid gas (generated by pouring nitric acid on copper filings, and collected over mercury). Result: Vac- cination was unsuccessful in each case, with disinfected points, and successful with the non- disinfected points from the same lot (S). Oil of Mustard, in the proportion of 1 : 33,000, prevents the development of the spores of bacilli (Koch.) The development of bacteria in unboiled beef-tea is prevented by 1 : 3,353 ; and 1 : 40 de- stroys the vitality of bacteria in broken-down beef- tea (de la Croix). Oil of Turpentine destroys the spores of bacilli in five days, and retards their development in the proportion of 1 : 75,000 (Koch). Turpentine has no action upon the virus of symptomatic anthrax (Arloing, Cornevin, and Thomas). Osmic Acid, in one per cent solution, destroys the spores of bacilli in one day (Koch). Oxalic Acid, in saturated solution, destroyed the virulence of the fresh virus of symptomatic an- thrax, but had no effect upon dried virus (Arloing, Cornevin, and Thomas). GERMICIDES AND ANTISEPTICS. 227 Ozone impairs, and, if maintained long in con- tact, destroys the activity of vaccine lymph (Braid- wood and Vacher). All germs suspended in the air, capable of developing in solutions of yeast from beer, are killed by ozone (Chappuis). Oxygen. — The experiments of Pasteur upon the attenuation of virus show that long exposure to the oxygen of the atmosphere reduces the repro- ductive activity of the micrococcus of fowl-cholera and of the anthrax bacillus, and that after a time the vitality of these organisms is destroyed. The spores of the anthrax bacillus are, however, un- affected by prolonged exposure. Out of twelve experimental vaccinations with vaccine exposed to oxygen (time of exposure one to seven days), but one was successful, and in this case there is reason to believe that the exposure was imperfect (Braid- wood and Vacher). Picric Acid prevents the development of the spores of bacilli in the proportion of 1 : 5,000 (Koch). The development of bacteria in beef- infusion is prevented by 1 : 2,005, and the bacteria of broken-down beef-tea are destroyed by 1 : 100 (de la Croix). Potash. — Caustic potash in the proportion of two per cent was fatal to the micrococcus of sep- ticaemia in one experiment, and failed in another ; eight per cent failed to kill the micrococcus of pus, while ten per cent was successful ; ten per cent failed to destroy the bacteria in broken-down beef- tea, and twenty per cent was successful (S). " Caus- 228 GERMICIDES AND ANTISEPTICS. tic potash has the minimum of preventive power, — 1 : 10. Such a mixture is highly caustic ; still it was necessary to use it of such strength as at 1 : 25 vibriones and bacteria were abundant " (Dougall). Potassium Arsenite (Fowler's solution of) failed to destroy the micrococcus of pus in the proportion of forty per cent. According to Koch, arsenite of potash prevents the development of anthrax spores in the proportion of 1 : 10,000. Potassium Chlorate in the proportion of four per cent does not destroy the virulence of septicoemic blood (S). " Chlorate of potassium, so much used as a gargle in stomatitis, diphtheria, etc., where it is held to act by destroying certain fungi or germs of specific poison, has not only no preventive power, but actually accelerates decomposition " (Dougall). Potassium Iodide gave no evidence of germicide power ; exposure to the action of a saturated solu- tion did not prevent the development of the bac- teria of broken-down beef-tea (S). Potassium Nitrate failed in 4 per cent solution to destroy the virulence of septicoBmic blood (S). Potassium Permanganate. — A 2 per cent solution destroys the virulence of septicoemic blood. The micrococcus of pus is destroyed by 1 : 800 (S). A 5 per cent solution destroys the fresh virus of symptomatic anthrax, but has no effect upon the dried virus (Arloing, Cornevin, and Thomas). One per cent will not destroy the spores of anthrax, GERMICIDES AND ANTISEPTICS. 229 but in the proportion of 1 : 3,000 their develop- ment is retarded (Koch). One part in three hun- dred prevents the development of bacteria in unboiled beef-infusion; and one part in thirty- five kills the bacteria of broken-down beef-tea (de la Croix). Pyrogallic Add. — A solution of one or two per cent prevents, for some months, the development of odors and of microscopic organisms ; a solu- tion of 2.5 per cent removes the odor from fluids in a state of putrefaction, and destroys bacteria (Bovet). Pyroligneous Add destroys the spores of the An- thrax bacillus in two days (Koch). Quinine. — A 10 per cent solution of sulphate of quinine has no action upon the bacterium of symp- tomatic anthrax (Arloing, Cornevin, and Thomas). One per cent of quinine, dissolved ivith muriatic add, destroys the spores of bacilli after ten days' ex- posure (Koch). The development of bacteria in a culture-fluid inoculated with a drop of turbid fluid from malarial soil is prevented by a solution of muriate of quinine of 1 : 900. From 1 : 1,000 to 1 : 1,500 non-putrid development begins. In a gelatine-culture from malarial soil no development occurred in solutions containing 1 : 1,500 ; non- putrid development occurred from 1 : 2,000 up to 1 : 3,000 ; and the development was accompanied by putrefaction when less than 1 : 9,000 was used (Ceri). Salicylic Add. — In the writer's experiments, this 230 GERMICIDES AND ANTISEPTICS. reagent was dissolved by means of sodium bibo- rate, which, by itself, in saturated solution, has no germicide power. A two per cent solution was found to destroy the micrococcus of pus and B. termo in active growth ; 4 per cent failed to destroy the bacteria in broken-down beef-tea (old stock). In the proportion of 1 : 200, this solution prevented the development of the micrococcus mentioned ; in 1 : 800, that of B. termo; and the septic micro- coccus in 1 : 400. But the antiseptic power exhib- ited by these figures does not differ from that obtained by the use of the solvent employed when used alone. The virus of symptomatic anthrax is destroyed by forty-eight hours' exposure to a solu- tion of salicylic acid of 1 : 1,000, and by saturated salicylic alcohol (Arloing, Cornevin, and Thomas). Salicylic acid dissolved in oil and in alcohol, in 5 per cent solution, does not destroy the spores of the anthrax bacillus (Koch). 1 : 200 destroys the bac- teria of sour milk (Molke). 1 : 343 killed the bacteria of beef-tea, and 1 : 1,121 prevented the de- velopment of bacteria in unboiled meat-infusion exposed to the air (de la Croix). The bacteria of tobacco-infusion were destroyed by 1 : 362, and their multiplication prevented by 1 : 932 (Bucholz). 1 : 724 prevented the development of bacteria in a vegetable infusion, and 1 : 1,000 in a solution of egg-albumen (Kiihn). Soda. — Caustic soda destroys the virulence of septicaBmic blood in the proportion of 1 : 400 (S). A one-in-five solution of soda destroys the virus GERMICIDES AND ANTISEPTICS. 231 of symptomatic anthrax when fresh, but has no effect upon dried virus (Arloing, Cornevin, and Thomas). Sodium Biborate. — The results obtained with this salt correspond, in the writer's experiments, with those given by boric acid. The virulence of septic blood, as shown by inoculation of rabbits, was destroyed by 2.5 per cent, while 1.25 per cent failed. That this is not due to germicide power is shown by the fact that a saturated solution does not kill the septic micrococcus, as proved by culture-experiments. It also failed with B. termo and the M. of pus. The multiplication of all of these organisms was, however, prevented by the presence of 1 : 200 in a culture-fluid, and B. termo failed to multiply in the presence of 1 : 400 (S). A 20 per cent solution does not destroy the viru- lence of the virus of symptomatic anthrax, as proved by inoculation experiments (Arloing, Cornevin, and Thomas). In the proportion of 1 : 107, the development of bacteria in unboiled beef-infusion is prevented, while 1 : 161 failed. 1 : 12 failed to kill the bacteria in broken down beef-tea (de la Croix). Sodium Chloride, in 5 per cent solution, failed to destroy the virulence of septicsemic blood .(S). Common salt ranks low as a preventive (Dougall). It is well known that meat may become putrid in a weak solution of brine, but the extended use of salt as a preservative agent demonstrates its anti- septic power, when used in a sufficiently strong 232 GERMICIDES AND ANTISEPTICS. solution. It is doubtful, however, whether infec- tious disease germs (spores) would be destroyed by the most concentrated solution. " A saturated solution of cloride of sodium did not destroy the virus of symptomatic anthrax in forty-eight hours' contact " (Arloing, Cornevin, and Thomas). Sodium Hyposulphite. — This salt, in the writer's experiments, gave no evidence whatever of germi- cide power. In saturated solution it failed to kill the bacteria in broken-down beef-tea, and the M. of pus was not destroyed by exposure for two hours to a thirty-two per cent solution. Nor was the development of the last-named organism pre- vented by the presence of this salt in a culture- solution in the proportion of eight per cent (S). Exposure for forty-eight hours to a fifty per cent solution does not destroy the virus of symptomatic anthrax (Arloing, Cornevin, and Thomas). Chloride of lime, hard soap, chloral um, and common salt are low preventives. The hyposulphite, borate, and sulphate of soda are useless as such (Dougall). Sodium Sulphite. — The results obtained corre- spond with those reported in the case of sodium hyposulphite (S.) Sodium Salicylate failed to destroy any of the test- organisms used in the writer's experiments, in the proportion of four per cent. But the virulence of septicsemic blood was destroyed by 1.25 per cent ; it must therefore have a restraining influence upon the development of the septic micrococcus, and doubtless upon other forms of bacteria also. GEEMICIDES AND ANTISEPTICS. 233 Sulphuric Acid destroys B. termo and the two species of micrococcus experimented upon in the proportion of 1 : 200 ; but a four per cent solution failed to destroy the bacteria in broken-down beef- tea (old stock), doubtless because of the presence of reproductive spores. The multiplication of the bacteria mentioned was prevented by the presence of this acid in a culture-solution, in the proportion of 1 : 800 (S). One part in 3,353 prevented the development of bacteria in unboiled meat-infusion, and 1 : 72 destroyed the bacteria of broken-down beef- tea (de la Croix). One part in 161 destroyed bacteria developed in tobacco-infusion (Bucholz). Sulphurous Acid. — Exp. No. 35, Jan. 15, 1880. — Five children were vaccinated from ivory points which had been exposed for six hours to an atmos- phere (dry) containing one per cent of sulphur dioxide (collected over mercury), and with five other points, from the same lot, not disinfected. Result : Vaccination was unsuccessful in each case with the disinfected points, and successful with those not disinfected (S). In four experiments in which dry vaccine was exposed to the fumes of sulphurous acid, for ten minutes, its infecting power was destroyed (Baxter). Sulphurous acid has no influence upon the bacteria of symptomatic anthrax (Arloing, Cornevin, and Thomas). It is powerless against the spores of the anthrax bacillus (Koch). In the proportion of 1 : 12,649, the de- velopment of bacteria in uncooked beef-infusion is prevented, and in 1 : 135 it destroys the vitality 234 GERMICIDES AND ANTISEPTICS. of the bacteria of broken-down beef-tea (de la Croix). Sulphuretted Hydrogen. — Bacteria develop read- ily in the presence of sulphuretted hydrogen (Hamlet). Tannic Acid, in the proportion of one per cent, destroys the virulence of septic blood (S). A one- iri-five solution of tannic acid has no effect upon the virus of symptomatic anthrax (Arloing, Corne- vin, and Thomas). A five per cent solution does not kill the spores of anthrax (Koch). Thymol dissolved in alcohol destroys the virulence of septicsemic blood (time of exposure half an hour), in the proportion of 1 : 400 (S). Thymol retards the development of anthrax spores in the proportion of 1 : 80,000 (Koch). One part in 200 kills the bacteria of tobacco-infusion (Bucholz) ; one part in 50 the sour-milk ferment (Molke) ; and one in 20 the bacteria of broken-down beef- tea (de la Croix). The development of bacteria in unboiled beef-infusion is prevented by 1 : 1,340 (de la Croix), and in Pasteur's fluid by 1 : 2,000 (Bucholz). Zinc Chloride destroys the micrococcus of gon- orrhoeal pus in the proportion of two per cent ; the septic micrococcus failed to multiply after ex- posure to one part in 200 (S). A five per cent solution failed within a month to weaken the de- veloping power of splenic fever spores (Koch). Liquor zinci chloridi (Squibbs) failed to kill the micrococcus of pus, in the proportion of eight per cent (S). GERMICIDES AND ANTISEPTICS. 235 Zinc Sulphate, in the proportion of twenty per cent, does not kill the micrococcus of pus ; but in the proportion of 1.25 per cent, it destroys the virulence of septicsemic blood. This is no doubt due to restraining power, and cannot be taken as evidence that the vitality of the septic micrococcus was destroyed (S). PART FIFTH. BACTERIA IN INFECTIOUS DISEASES. No more important question has ever engaged the attention of physicians, of sanitarians, or of biologists, than that which relates to the role of the bacteria in infectious diseases. The practical results of etiological studies, so far as the preven- tion and cure of disease are concerned, are likely to be much greater than those which have been gained by the pathologists ; and, if the time ever comes, as now seems not improbable, when we can say with confidence, infectious diseases are parasitic diseases, medicine will have established itself upon a scientific foundation. But this gener- alization, which some physicians think is justified, even now, by the experimental evidence which has been so rapidly accumulating during the past de- cade, would, in the opinion of the writer, be prem- ature in the present state of science. And, for the present, it seems wiser to encourage additional researches rather than to attempt to generalize from the data at hand. For much of the evidence offered in favor of this view is open to question ; BACTEEIA IN INFECTIOUS DISEASES. 237 and even where we do not doubt the scientific accuracy of an observer, we may differ from him as to the interpretation of the facts which he has recorded. Those who have had the most experi- ence in this difficult field of investigation, are commonly the most critical and exacting with reference to the alleged discoveries of others. And it is now generally admitted that the only satisfactory proof that a certain micro-organism bears a causal relation to a disease with which it is associated is that which is obtained by a series of culture experiments, in which the organism is completely isolated from the non-living constitu- ents of the infective material containing it, and in the production of the disease in question by inoculation experiments with such a " pure-cul- ture." The unimpeachable nature of this proof, when the experiment is properly made and fre- quently repeated with the same result, is made apparent in the following quotation from a paper by the writer relating to " a fatal form of septi- caemia in the rabbit." 1 " In my previous paper I related a series of experi- ments commenced July 6th, to which I must refer the reader as properly introducing the following : — " The culture-fluid (No. 6) used in Experiment No. 3 (July 26th) was laid aside in an hermetically-sealed culture-flask until September 12th, when a minute drop was used to inoculate sterilized bouillon in culture-tube No. 7. This, placed in a culture-oven at 100° Fahr. for twenty-four hours, became clouded, and upon micro- 1 Med. Times, Phila., Nov. 4th, 1882, p. 81. 238 BACTERIA IN INFECTIOUS DISEASES. scopical examination proved to be pervaded with the identical micrococcus heretofore described and photo- graphed (See Fig. 2, Plate IX.). A drop of culture No. 7 was in like manner used to inoculate culture No. 8, and the next day, this being also pervaded by the micrococcus, was used in the following experiment : — " Exp. No. 4. — September 14th. — Injected ten minims of culture No. 8 into a full-grown rabbit. Result : This animal died at 9 A. M. September 15th, and a micro- scopical examination made at once demonstrated the presence of the micrococcus in great numbers in the blood and in effused serum in the sub-cutaneous connec- tive tissue. u Remarks. — This experiment shows that the micro- coccus retained its vitality and its full virulence at the end of six weeks, and, very conclusively, that the viru- lence of the culture-fluid is due to the presence of the micrococcus, and not to a hypothetical chemical virus found in the first instance in human saliva and subse- quently in the blood of a rabbit inoculated with this fluid. For the benefit of those who have not calculated the degree of dilution which such a hypothetical chemi- cal virus would undergo in such a series of culture ex- periments, I submit the following simple calculation : — " My culture-tubes contain about a fluidrachm of sterilized bouillon. The amount of blood introduced into culture No. 1, as seed, was considerably less than a minim ; but for convenience I will suppose that one minim is used each time to start a new culture, — that is, the original material is diluted 60 times in the first cul- ture, 3,600 times in the second, 216,000 times in the third, and in the eighth culture it will be present in the proportion of one part in 1,679,611,600,000,000. Yet a few minims of this eighth culture possesses all the viru- lence of the first. BACTERIA IN INFECTIOUS DISEASES. 239 " Look at it from another point of view. The few minims of culture -fluid introduced beneath the skin of a rabbit contain a micrococcus presenting definite mor- phological characters. The blood of the animal which falls a victim to experimental inoculation with this fluid is filled within forty-eight hours with the same micro- organism in numbers far exceeding the normal histo- logical elements, — red and white corpuscles ; yet some very conservative physicians still claim that the invading parasite is without import, a mere epi-phenomenon, while the infinitesimal portion of a hypothetical chemi- cal virus is credited with this malignant potency." When, in addition to this, we remember that potent chemical poisons, especially when injected subcutaneously, act promptly, and that their poi- sonous effect bears a relation to the dose in which they are administered, whereas a rabbit subjected to an experimental inoculation with septic blood, or with a culture-fluid remotely inoculated with this material, shows no signs of ill-health for many hours, — eighteen hours or more, — and that it is only when sufficient time has elapsed to permit of the abundant development of the micrococcus that serious symptoms are developed, we shall see that but one conclusion can be drawn as regards the role of the micrococcus. It is by experimental evidence of this nature that Koch, Pasteur, and many others have demon- strated beyond question that the disease known as anthrax is produced by a parasitic micro-organism, — the Bacillus anthracis ; that the last-named in- vestigator has established the etiological role of the 240 BACTERIA IN INFECTIOUS DISEASES. micrococcus of fowl-cholera ; and that Koch has proved that a form of induced septicaemia in mice, which he has especially studied, is due to a minute bacillus. It has been suggested that the parasitic micro- organism in these diseases is, perhaps, only a second- ary cause, being merely a carrier of the non-living ferment, which is the special poison of the disease. This hypothesis, also, is excluded by inoculation experiments with a pure-culture, sufficiently re- moved from the natural infective material. For the organisms introduced into culture No. 1, as seed, disappear as quickly from successive cultures as does the non-living material with which they are associated, and we may very soon leave them out of the account, although each successive culture- fluid is invaded throughout by their numerous progeny. Having determined for a certain infectious dis- ease that its transmissibility depends upon the presence in the infective material of a living micro- organism, the question naturally arises as to the modus opemndi of this parasite. Does it produce death by appropriating something from the vital fluid, or from the tissues invaded by it, — e. g., oxy- gen, which is essential for the maintenance of vital processes in the living animal ? Or does it, at the same time that it appropriates material for its own nutrition, evolve some poisonous chemical product which is the immediate cause of the morbid pheno- mena in the infected animal ? Or does it produce BACTERIA IN INFECTIOUS DISEASES. 241 death by the mechanical effects which result from its presence in such vast numbers, i. e., by blocking up the capillaries and the formation of emboli ? There can be little doubt that, in these acute infectious diseases, the parasite injures its host in all three of the ways indicated, and that a fatal result is to be ascribed to the three causes men- tioned conjointly. A most difficult and important question in con- nection with these diseases is that which relates to the rationale of the immunity produced by protec- tive inoculations practised by one of the methods described in PART FOURTH of the present volume. In these protective vaccinations, the virus used is either greatly diluted or is modified as regards the reproductive activity of the parasite by exposure to oxygen, by heat, or by certain chemical re- agents. A susceptible animal, when inoculated with virus " attenuated " by one of these methods, does not succumb to the attacks of the parasite, but, after experiencing a mild form of the disease, recovers, and is subsequently protected from the effects of full doses of unmodified virus. This recovery after inoculation with attenuated virus is more easy to understand than is the subse- quent protection. There is evidently some pro- vision of nature by which invading organisms may be disposed of when they do not multiply too quickly, but which fails when they have very great reproductive activity, and when the con- ditions within the living animal are extremely 16 242 BACTERIA IN INFECTIOUS DISEASES. favorable to their development. These conditions doubtless relate mainly to the composition and temperature of the culture-medium — i.e., of the blood of the animal — and consequently vary in different species of animals. But that the compo- sition of the blood should be changed materially and permanently in the same animal, as a result of the mild form of disease which follows pro- tective inoculation, it is difficult to believe. Yet this is the explanation given by Pasteur of the immunity afforded by such inoculations. This change is supposed to consist in the removal of some material essential for the nutrition of the microbe, which is exhausted during the attack, and never reproduced. This view is sustained in the following language : — " It is the life of a parasite in the interior of the body which produces the malady commonly called ' cholera des poulesj and which causes death. From the moment when this culture (i. e., the multiplication of the para- site) is no longer possible in the fowl, the sickness can- not appear. The fowls are then in the constitutional state of fowls not subject to be attacked by the disease. These last are as if vaccinated from birth for this mal- ady, because the foetal evolution has not introduced into their bodies the material necessary to support the life of the microbe ; or these nutritive materials have disap- peared at an early age. " Certainly one should not be surprised that there may be constitutions sometimes susceptible and some- times rebellious to inoculation — that is to say, to the cultivation of a certain virus, when, as I have an- BACTERIA IN INFECTIOUS DISEASES. 243 nounced in my first note, one sees a preparation of beer yeast made exactly like one from the muscles of fowls (bouillon) to show itself absolutely unsuited for the cultivation of the parasite of fowl cholera, while it is admirably adapted to the cultivation of a multitude of microscopic species, notably to the bacteride charbon- neuse (Bacillus anthracis). " The explanation to which these facts conduct us, as well of the constitutional resistance of some individuals, as of the immunity produced by protective inoculations, is only natural when we consider that every culture, in general, modifies the medium in which it is effected ; a modification of the soil when it relates to ordinary- plants; a modification of plants and animals when it relates to their parasites ; a modification of our culture liquids when it relates to mucedines, vibrioniens, or ferments. " These modifications are manifested and character- ized by the circumstance that new cultivations of the same species in these media become promptly difficult or impossible. If we sow chicken-bouillon with the mi- crobe of fowl-cholera, arid, after three or four days, filter the liquid in order to remove all trace of the microbe, and subsequently sow anew in the filtered liquid this parasite, it will be found quite powerless to resume the most feeble development. The liquid, which is perfectly limpid after being filtered, retains its limpid- ity indefinitely. " How can we fail to believe that by cultivation in the fowl of the attenuated virus, we place its body in the state of this filtered liquid, which can no longer cultivate the microbe ? The comparison can be pushed still further ; for, if we filter the bouillon containing the microbe in full development, not on the fourth day of culture, but on the second, the filtered liquid will still 244 BACTERIA IN INFECTIOUS DISEASES. be able to support the development of the microbe, although with less energy than at the outset. We comprehend, then, that after a cultivation of the modi- fied (attenue) microbe in the body of the fowl, we may not have removed from all parts of its body the aliment of the microbe. That which remains will permit, then, a new culture, but in a more restricted measure. " This is the effect of a first inoculation ; subsequent inoculations will remove progressively all the material necessary for the development of the parasite. " Is this the only possible explanation of the phenom- enon ? No ; we may admit the possibility that the development of the microbe, in place of removing or destroying certain matters in the bodies of the fowls, adds, on the contrary, something which is an obstacle to the future development of this microbe. The history of the life of inferior beings authorizes such a supposi- tion. The excretions resulting from vital processes may arrest vital processes of the same nature. In cer- tain fermentations we see antiseptic products make their appearance during, and as a result of, the fermen- tation, which put an end to the active life of the fer- ments, and arrest the fermentations long before they are completed. In the cultivation of our microbe, pro- ducts may have been formed the presence of which, possibly, may explain the protection following inocula- tion. " Our artificial cultures permit us to test the truth of this hypothesis. Let us prepare an artificial culture of the microbe, and after having evaporated it, in vacuo, without heat, let us bring it back to its original volume by means of fresh chicken bouillon. If the extract con- tains a poison for the life of the microbe, and if this is the cause of its failure to multiply in the filtered liquid, the new liquid should remain sterile. Now this BACTERIA IN INFECTIOUS DISEASES. 245 is not the case. We cannot, then, believe that during the life of the parasite certain substances are produced which are capable of arresting its ulterior develop- ment." — (Comptes rendus Acad. des &?., XC. pp. 952- 958.) It is a little surprising that after disproving, by the experimental method, the hypothesis last men- tioned, which had been proposed by a member of the French Academy in explanation of the phe- nomenon in question, Pasteur did not, in accord- ance with his usual custom, attempt to establish his own hypothesis upon a firm foundation by an experiment which at once suggests itself. If a fowl which is protected against cholera, or an animal which is protected against anthrax, owes this protection to the fact that a certain material which is required for the development of the microbe of fowl-cholera, or for the anthrax bacil- lus, has been exhausted in the course of the modi- fied form of the disease to which immunity is due, then the flesh of such an animal, made into bouillon, should not constitute a proper culture-medium for the organisms in question. The writer ventures to predict that the result of such an experiment would not be favorable to Pasteur's hypothesis, and that it will be found that the micrococcus of fowl-cholera can be cultivated in bouillon made from the flesh of a protected animal, and that the bacil- lus of anthrax may multiply freely in the blood, or in an infusion of the flesh, of an animal which, before it was killed for the experiment, possessed 246 BACTERIA IN INFECTIOUS DISEASES. immunity against the disease anthrax. The writer long since proposed to himself to make the experi- ment, but has not yet been able to do so. The matter is mentioned here in the hope that some one more favorably situated for pursuing experi- mental work will consider it of sufficient impor- tance to induce him to test it in the manner indicated. In the meantime I take the liberty of quoting, from a paper published in 1881, certain extracts in which my reasons are given for doubt- ing the correctness of the hypothesis of Pasteur, and in which another explanation is offered : — " Let us see where this hypothesis leads us. In the first place, \ve must have a material of small-pox, and a material of measles, and a material of scarlet fever, etc., etc. Then we must admit that each of these different materials has been formed in the system and stored up for these emergencies, — attacks of the diseases in ques- tion,— for we can scarcely conceive that they were all packed away in the germ-cell of the mother and the sperm-cell of the father of each susceptible individual. If, then, these peculiar materials have been formed and stored up during the development of the individual, how are we to account for the fact that no new produc- tion takes place after an attack of any one of the dis- eases in question ? "Again, how shall we account for the fact that the amount of material which would nourish the small-pox germ, to the extent of producing a case of confluent small-pox may be exhausted by the action of the atten- uated virus (germ) introduced by vaccination ? Pas- teur's comparison of a fowl protected by inoculation with the microbe of fowl-cholera, with a culture-fluid in BACTERIA IN INFECTIOUS DISEASES. 247 which the growth of a particular organism has exhausted the pabulum necessary for the development of additional organisms of the same kind, does not seem to me to be a just one, as in the latter case we have a limited supply of nutriment, while in the former we have new supplies constantly provided of the material — food — from which the whole body, including the hypothetical substance essential to the development of the disease-germ, was built up prior to the attack. Besides this, we have a constant provision for the elimination of effete and useless products. " This hypothesis, then, requires the formation in the human body, and the retention up to a certain time, of a variety of materials, which, so far as we can see, serve no purpose except to nourish the germs of various spe- cific diseases, and which, having served this purpose, are not again formed in the same system, subjected to simi- lar external conditions, and sup plied with the same kind of nutriment. "The difficulties into which this hypothesis leads certainly justify us in looking further for an explanation of the phenomenon in question. This explanation is, I believe, to be found in the peculiar properties of the protoplasm, which is the essential frame-work of every living organism. The properties referred to are : the tolerance which living protoplasm may acquire to cer- tain agents which, in the first instance, have an inju- rious or even fatal influence upon its vital activity, and the property which it possesses of transmitting its pecu- liar qualities, inherent or acquired, through numerous generations, to its offshoots or progeny. " Protoplasm is the essential living portion of the cel- lular elements of animal and vegetable tissues ; but as our microscopical analysis of the tissues has not gone beyond the cells of which they are composed, and is not 248 BACTERIA IN INFECTIOUS DISEASES. likely to reveal to us the complicated molecular struc- ture of the protoplasm upon which, possibly, the proper- ties under consideration depend, it will be best, for the present, to limit ourselves to a consideration of the living cells of the body. These cells are the direct descend- ants of pre-existing cells, and may all be traced back to the sperm-cell and germ-cell of the parents. Now, the view which I am endeavoring to elucidate is, that dur- ing a non-fatal attack of one of the specific diseases, the cellular elements implicated, which do not succumb to the destructive influence of the poison, acquire a tol- erance to this poison which is transmissible to their progeny, and which is the reason of the exemption which the individual enjoys from future attacks of the same disease. 4' The known facts in regard to the hereditary trans- mission, by cells, of acquired properties, make it easy to believe in the transmission of such a tolerance as we imagine to be acquired during the attack ; and if it is shown by analogy that there is nothing improbable in the hypothesis that such a tolerance is acquired, we shall have a rational explanation, not of heredity and the mysterious properties of protoplasm, but of the partic- ular result under consideration. The transmission of acquired properties is shown in the budding and graft- ing of choice fruits and flowers, produced by cultiva- tion, upon the wild stock from which they originated. The acquired properties are transmitted indefinitely ; and the same sap which on one twig nourishes a sour crab-apple, on another one of the same branch is elab- orated into a delicious pippin. . . " The tolerance to narcotics — opium and tobacco, and to corrosive poisons — arsenic, wrhich results from a gradual increase of dose, may be cited as an example of acquired tolerance by living protoplasm to poisons, BACTERIA IN INFECTIOUS DISEASES. 249 which at the outset would have been fatal in much smaller doses. " The immunity which an individual enjoys from any particular disease must be looked upon as a power of resistance possessed by the cellular elements of those tissues of his body which would yield to the influence of the poison in the case of an unprotected person. . . . The resistance of living matter to certain destructive influences is a property depending upon vitality. Thus, living protoplasm resists the action of the bacteria of putrefaction, while dead protoplasm quickly undergoes putrefactive changes." — Am. J. of the Med. Sciences, April, 1881,|?. 375. The hypothesis of Pasteur would account for the fact that one individual suffers a severe attack and another a mild attack of an infectious disease, after being subjected to the influence of the poison under identical circumstances, by the supposition that the pabulum required for the development of this particular poison is more abundant in the body of one individual than in the other. The expla- nation which seems to us more satisfactory, is that the vital resistance offered by the cellular elements in the bodies of these two individuals was not the same for this poison. It is well known that in conditions of lowered vitality, resulting from star- vation, profuse discharges, or any other cause, the power to resist disease-poisons is greatly dimin- ished, and, consequently, that the susceptibility of the same individual differs at different times. From our point of view, the blood, as it is found within the vessels of a living animal, is not simply 250 BACTERIA IN INFECTIOUS DISEASES. a culture-fluid maintained at a fixed temperature ; but, under these circumstances, is a tissue, the histological elements of which present a certain vital resistance to pathogenic organisms which may be introduced into the circulation. If we add a small quantity of a culture-fluid containing the bacteria of putrefaction to the blood of an animal, withdrawn from the circulation into a proper receptacle, and maintained in a culture- oven at blood-heat, we will find that these bacteria multiply abundantly, and evidence of putrefactive decomposition will soon be perceived. But, if we inject a like quantity of the culture-fluid with its contained bacteria into the circulation of a living animal, not only does no increase and no putrefac- tive change occur, but the bacteria introduced quickly disappear, and at the end of an hour or two the most careful microscopical examination will not reveal the presence of a single bacterium. This difference we ascribe to the vital properties of the fluid as contained in the vessels of a living animal ; and it seems probable that the little masses of protoplasm known as white blood cor- puscles are the essential histological elements of the fluid, so far as any manifestation of vitality is concerned. The writer has elsewhere suggested that the disappearance of the bacteria from the circulation, in the experiment above referred to, may be effected by the white corpuscles, which, it is well known, pick up, after the manner of amoebce, any BACTERIA IN INFECTIOUS DISEASES. 251 particles, organic or inorganic, which come in their way. And it requires no great stretch of credulity to believe that they may, like an amoeba, digest and assimilate the protoplasm of the captured bac- terium, thus putting an end to the possibility of its doing any harm. In the case of a pathogenic organism we may imagine that, when captured in this way, it may share a like fate if the captor is not paralyzed by some potent poison evolved by it, or overwhelmed by its superior vigor and rapid multiplication. In the latter event, the active career of our conser- vative white corpuscle would be quickly termin- ated, and its protoplasm would serve as food for the enemy. It is evident that in a contest of this kind the balance of power would depend upon cir- cumstances relating to the inherited vital charac- teristics of the invading parasite and of the in- vaded leucocyte. That different pathogenic organisms of the same species may differ as to their power to overcome the vital resistance of living animals is amply proved by experiment. We have examples of this in the attenuated virus of anthrax and of fowl- cholera. These physiological varieties, as Pasteur calls them, may be produced at will by one of the methods heretofore referred to. They differ from the unmodified virus in vital activity, and this is especially manifested in their diminished reproduc- tive power. In the great laboratory of nature, like causes 252 BACTERIA IN INFECTIOUS DISEASES. must produce similar results; and there can be little doubt that physiological varieties, or breeds, of the different species of bacteria are constantly being produced and destroyed by the operation of natural causes. Under the influence of a favorable temperature and of abundant pabulum, these mi- nute plants multiply abundantly ; and, in accord- ance with the laws of natural selection, there must be a constant tendency among them to develop those characters which are most favorable to their preservation, — e. g., a capacity for rapid multi- plication, and to adapt themselves to their envi- ronment. If we suppose that under certain circumstances the conditions relating to environment approach those which would be found within the body of a living animal, we can easily understand how a micro-organism, which has adapted itself to these conditions, may become a pathogenic organism, when by any chance it is introduced into the cir- culation of such an animal. The culture-fluid — blood — and temperature being favorable, it is only a question of superiority by vital resistance on the one hand, or by reproductive activity on the other. That harmless species of bacteria may develop pathogenic properties in the manner indicated, seems extremely probable ; and we should a priori expect that such a result would occur more frequently in the tropics, where the elevated temperature and abundance of organic pabulum BACTERIA IN INFECTIOUS DISEASES. 253 furnish the favorable conditions required. In this way we may, perhaps, explain the origin of epi- demics of pestilential diseases, such as yellow fever and cholera. If these diseases do not, at the pres- ent day, originate in the manner indicated, they at all events have their permanent abiding place in tropical countries. Although the specific germs of these diseases have not been demonstrated, there is strong reason for believing that they re- sult from the direct or indirect action of living ferments. For there is abundant evidence to prove that the specific poisons to which they are due may multiply indefinitely external to the bodies of the sick. Such multiplication is a property of liv- ing matter only. Moreover, the conditions which favor this multiplication — an elevated tempera- ture and the presence of decomposing organic material — are exactly the conditions required for the development of low organisms. The experimental transformation of the harm- less hay-bacillus (B. subtilis) into the deadly Ba- cillus anthracis has been claimed by Buchner and by Nageli ; and Prof. Greenfield claims to have transformed, by a series of culture experiments, the anthrax bacillus into a harmless form not dis- tinguishable from the hay bacillus. Koch insists, however, that these are distinct species, and the weight of evidence seems to be in favor of this view. However this may be, it is beyond ques- tion that the anthrax bacillus may undergo a re- markable modification as regards virulence; and 254 BACTERIA IN INFECTIOUS DISEASES. Pasteur asserts that this virulence may be restored by inoculating guinea-pigs but a day old, which succumb to this attenuated virus, although those which are five or six days old are proof agains it. After several successive inoculations, older guinea- pigs are killed, and after a time the virus becomes sufficiently potent to destroy a full-grown animal. Finally it regains its full activity, and will kill a sheep. The form of induced septicaemia in the rabbit, which has been especially studied by the writer (see pt 354), furnishes a good example of an in- fectious disease resulting in one species of animal from the introduction into its body of a micro- organism which is harmless for other species. This organism — a micrococcus — is commonly found in normal human saliva, where it is asso- ciated with various other species. Experiments thus far made indicate that there are various physiological varieties (breeds) of this micrococ- cus, varying in pathogenic power ; for the saliva of different individuals differs in virulence. This may be accounted for by the fact that the con- ditions are not identical. The human mouth is a culture-apparatus in which the conditions are ex- tremely favorable for the development of these minute plants ; the secretions from the salivary glands afford a constant supply of pabulum, and the temperature is maintained at a fixed point. But the flow of saliva is more abundant in some persons than in others; and the presence of de- BACTERIA IN INFECTIOUS DISEASES. 255 cayed teeth and of organic material, from neglect of the tooth-brush, may favor the development of putrefactive bacteria, which are fatal to the spe- cies of micrococcus which produces septicaemia in rabbits. Differences in habit as to the expectora- tion of the saliva or retaining it in the oral cavity, and as to breathing through the nose or through the mouth, will also constitute differences in the environment of the micrococcus which can scarcely fail to have an influence upon its physiological characters. When the flow of saliva is rapid, and it is not long retained in the mouth, it is evident that an organism which multiplies rapidly will have the advantage of one which multiplies slowly and may survive where the other would quickly disappear. There will also be a constant ten- dency to develop still further this capacity for rapid multiplication, which no doubt is an impor- tant, if not the essential, factor in giving to a micro-organism pathogenic power. The impor- tance of this factor will be appreciated when we remember that one method by which nature limits the power for mischief of putrefactive bacteria injected into the tissues is by a conservative inflammatory process, which builds a wall about the invading parasites, and confines their depreda- tions within the narrow limits of an abscess. In the disease produced by inoculation with saliva, or with a culture-fluid containing the micrococcus under consideration, owing perhaps to the rapid development of the micrococcus, no such limiting 256 BACTERIA IN INFECTIOUS DISEASES. wall of inflammatory exudation is established ; and we find the subcutaneous connective tissue diffusely infiltrated with serum which swarms with the parasite. The failure to restrict the inroads of the para- site may not be due alone to its power of rapid multiplication. It is not improbable that some poison is produced, during its active growth, which lowers the vital resistance of the tissues and pre- vents the occurrence of conservative adhesive in- flammation. And it may be that the true expla- nation of the immunity afforded by a mild attack of an infectious germ-disease is to be found in an acquired tolerance to the action of a chemical poi- son produced by the micro-organism, and conse- quent ability to bring the resources of nature to bear to restrict invasion by the parasite. In the infectious disease known as hospital gan- grene, circumstances relating to the origin, nature, and treatment of the malady make it seem ex- tremely probable that some species of bacterium, ordinarily harmless, develops pathogenic proper- ties as the result of an unusually favorable environ- ment, and becomes the infecting agent, which, by invading the enfeebled tissues, causes the rapidly extending necrosis which is characteristic of this frightful malady. This disease is developed de novo in the surgical wards of hospitals, where numerous patients, with profusely discharging wounds, are brought together. Like its congeners, erysipelas and puerperal fever, it is prevented by cleanliness BACTERIA IN INFECTIOUS DISEASES. 257 and antiseptic treatment. Its progress cannot, however, be arrested by ordinary antiseptic appli- cations ; for the pathogenic organism (hypothetical as yet) invades the tissues to a certain depth, and its destruction requires something more than a superficial germicide action, — e. g., bromine, nitric acid, the hot iron. Diphtheria, also, is a disease in which there seems to be good reason for believing that the different degrees of virulence are due to circumstances re- lating to the genealogy of the infecting organism, as well as to the resisting power of the infected individual ; and that, as in anthrax and in fowl- cholera, physiological varieties of the pathogenic micrococcus, to which this disease is probably due, may be developed by special conditions relating to its environment, either in the fauces of an infected individual or external to the human body. It is not alone by invading the blood or tissues that bacteria exhibit pathogenic power. Chemical products evolved during their vital activity, ex- ternal to the body, or in abscesses and suppurating wounds, or in the alimentary canal, may doubtless be absorbed and exercise an injurious effect upon the animal economy. Indeed, we have experi- mental evidence that most potent poisons are pro- duced during the putrefactive decomposition of organic matter. The poisons, resembling the vege- table alkaloids in their reactions, called ptomaines by Selmi, who first obtained them from a cadaver, are fatal to animals in extremely minute doses. 17 258 BACTERIA IN INFECTIOUS DISEASES. These ptomaines have also been obtained by Gau- tier from putrid blood and from the normal secre- tions of healthy persons, — saliva, urine, blood, etc. The soluble poison, sepsin, which has been shown by the researches of Bergmann, Panum, Burdon Sanderson, and others, to exist in putrid blood is fatal to animals when administered in a sufficient dose, which, however, is very small. According to Koch five drops of blood, which has not putrefied too long, is sufficient to kill a mouse within a short time. After receiving an injection of this kind the symptoms of poisoning are de- veloped immediately, and the animal dies in from four to eight hours. " In such a case the greater part of the fluid injected is found in the subcutaneous cellular tissue of the back in much the same condition as before it was injected. It contains bacteria of the most diverse forms, irregularly mixed together, and as numerous as when examined before injection. No inflammation can be observed in the neighborhood of the place of injection. The inter- nal organs are also unaltered. If blood taken from the right auricle be introduced into another mouse, no effect is produced. Bacteria cannot be found in any of the internal organs or in the blood of the heart. " An infective disease has therefore not been produced as the result of the injection. On the other hand, there can be no doubt that the death of the animal was due to the soluble poison, sepsin. " This supposition is confirmed by the fact that when BACTERIA IN INFECTIOUS DISEASES. 259 less fluid is introduced into the animal the symptoms of poisoning which follow are less marked, and are quite absent when one or at most two drops have been in- jected." l On the otlier hand, the infectious disease which results in certain cases from a similar inoculation produces death only at the end of forty to sixty hours, and is attended with definite pathological lesions and the presence of a minute bacillus in the blood and tissues of the infected animal. A very small quantity (e. g., one-tenth of a drop) of the fluid of the subcutaneous oedema, or of blood from the heart of such an animal, is sufficient to infect another, and Koch has fully demonstrated the infectious nature of the disease by a series of seventeen successive inoculations. He says : " It is sufficient, in order to bring about the death of the animal in about fifty hours, to pass the point of a small scalpel, which has been in contact with the infected blood, over a small wound in the skin." This distinction between septic toxaemia and in- fectious septicaemia, which has been established by the experimental researches of Koch, Pasteur, and many others, is opposed to the results reported by Rosenberger of Wurzburg, who claims to have demonstrated that the various forms of septic micro-organisms appear in the body of an animal which has been subjected to experimental inocula- tion, not because like organisms have been intro- 1 Traumatic Infectious Diseases, Sydenham Society's translation, London, 1880, p. 35. 260 BACTERIA IN INFECTIOUS DISEASES. duced as seed, but as a result of the introduction of a chemical poison which causes organisms pre- viously present in the body of the animal to make their appearance in the blood, etc. That the injection of sepsin favors the develop- ment of bacteria introduced at the same time is very probable, and we cannot help believing that Rosenberger has unwittingly introduced living bacteria with his cooked septic blood and serum, notwithstanding the precautions which he claims to have taken. This view is supported by the ex- periments of Zuelzer and Sonnenschein, who, finding a resemblance between the physiological effects of sepsin and of atropia, injected two to five centigrammes of neutral sulphate of atropia at the same time with a culture-solution contain- ing bacteria. Fatal septicaemia was found to re- sult from these inoculations, while the bacteria injected alone did no harm. The subcutaneous injection of other potent poi- sons has been found to be followed by local necro- sis and rapidly developed putrefactive changes ; but there is reason to believe that in . these in- stances, also, the putrefactive germs are intro- duced simultaneously with the chemical poison, or find their way through the inoculation wound from the exterior, rather than to suppose that they are developed within the body of the animal. For the observations and experiments of numerous investigators are opposed to the belief that bacte- ria are habitually present in the blood and tissues BACTERIA IN INFECTIOUS DISEASES. 261 of living animals. They are known to infest the alimentary canal, and it is probable that the small- est portion of hair or epithelium detached from the surface of the body qf any one of the lower animals would fertilize a culture-solution ; but blood drawn from the veins with proper precau- tions does not fertilize a sterilized culture-solution. Koch says (I. . These bacilli were found in one case only . INTERMITTENT FEVER. 321 sisted of culture-experiments with organisms found in malarial and other soils, of experiments on animals, and culture-experiments with quinine. They resulted in proving that the spores could be cultivated, — Ceri applying the term natural germs to those found in the atmosphere and soil, and artificial germs to those which result from their culture ; that animals could be infected by their injection into the blood, though to a less degree by the cultivated than by the natural germs, the former growing weaker in successive generations ; and that the infecting properties could be retarded by the appli- cation of heat to culture-fluids, and the introduction of quinine into them, certain degrees of the former and strengths (1: 800) of the latter making the culture of the spores impossible, and arresting the putrid fer- mentation induced by them. The practical application of these facts is self-evident. " Finally the opportunity has recently been present- ed to Dr. Franz Ziehl to test these results clinically 1 in three typical cases of malaria, in all of which the spleen was enlarged. In all three the bacilli above de- scribed were found in the blood taken from any part of the body by the prick of a needle, and examined in the fresh state, or dried in a thin layer upon a cover-glass, by simply passing the latter over a flame. These have been preserved by Dr. Ziehl for three months without undergoing any change. " The bacilli thus observed were of different lengths, but usually were from one-fourth to the entire diameter of a red corpuscle. The majority of those measured were about 4 micro-millimetres long and .7 broad. Their ends were swollen and roundish." The evidence as here stated certainly seems very i 1 Deutsche medizinische Wochensehrift, Nov. 25, 1882. 21 322 BACTERIA IN INFECTIOUS DISEASES. complete, and the writer freely admits that the nega- tive results which he report- ed after an attempt to repeat the experiments of Klebs and Tommasi-Crudeli, made under the auspices of the National Fig 15 Board of Health in New Or- The Bacillus malaria, as seen l™™> du™S tllG SUinmer of in blood obtained from a pa- 1880, CaUllOt be given ffFCat tient seized with intermittent _ ° fever, taken during the chill. WClght aS OppOSed tO thcSC (From Tommasi-Crudeli.) .,. ^ positive statements. But the fact that no confirmation has yet come from Eng- lish or American sources during the time which has elapsed since the discovery of Klebs and Tommasi-Crudeli was announced, constitutes nega- tive evidence of a much stronger character. The Bacillus malar ice, according to all accounts, should be much easier to recognize than Koch's bacillus of tuberculosis, which has already been seen by numer- ous physicians in nearly every large city in this country and in Europe. But who on this side of the Atlantic has seen the Bacillus inalamc? Yet malarial fevers are widespread, and a microscope is to be found in nearly every physician's office. The writer has searched faithfully for this bacillus in the blood of patients in the Charity Hospital, New Orleans, selected for him by Professor Beiniss as well-marked cases of malarial fever, but admits that he has not sought it in blood from the spleen, or in that drawn during a chill, except in two or INTERMITTENT FEVER. 323 three instances. He is therefore anxious to make more extended researches whenever the opportu- nity may offer, and will not fail to report promptly any future observations more in correspondence with those of the German and Italian investigators named. In the report of the experimental investigation referred to, the following summary statement is made : — " Among the organisms found upon the surface of swamp-mud, near New Orleans, and in the gutters within the city limits, are some which closely resemble, and perhaps are identical with, the Bacillus malarice of Klebs and Tommasi-Crucleli ; but there is no satisfactory evidence that these or any of the other bacterial organ- isms found in such situations, when injected beneath the skin of a rabbit, give rise to a malarial fever correspond- ing with the ordinary paludal fevers to which man. is subject. " The evidence upon which Klebs and Tommasi-Cru- deli have based their claim of the discovery of a Bacillus malarice cannot be accepted as sufficient ; («) because in their experiments and in my own the temperature curve in the rabbits experimented upon has in no case exhibited a marked and distinctive paroxysmal char- acter ; (6) because healthy rabbits sometimes exhibit diurnal variations of temperature (resulting apparently from changes in the external temperature) as marked as those shown in their charts ; (c) because changes in the spleen such as they describe are not evidence of death from malarial fever, inasmuch as similar changes occur in the spleens of rabbits dead from septicaemia produced by the subcutaneous injection of human saliva; 324 BACTERIA IN INFECTIOUS DISEASES. (c?) because the presence of dark-colored pigment in the spleen of a rabbit cannot be taken as evidence of death from malarial fever, inasmuch as this is frequently found in the spleens of septicsemic rabbits. " While, however, the evidence upon which Klebs and Tommasi-Crudeli have based their claim to a dis- covery is not satisfactory, and their conclusions are shown not to be well founded, there is nothing in my researches to indicate that the so-called Bacillus malarice, or some other of the minute organisms associated with it, is not the active agent in the causation of malarial fevers in man. On the other hand, there are many cir- cumstances in favor of the hypothesis that the etiology of these fevers is connected, directly or indirectly, with the presence of these organisms or their germs in the air and water of malarial localities." It will be seen that I am not able to agree with the editorial above quoted in the statement, that " the animals infected by them" — i. e., the spores of Bacillus mulnriw — " exhibited not only the clinical course of malarial disease as seen in man, but also the post mortem appearances." On the other hand, I do not find in the temperature-charts published by Klebs and Tommasi-Crudeli in their original report, and copied by me in my report referred to, satisfactory evidence of the production of a fever characterized by regularly recurring paroxysms, like the ordinary paludal fevers in man ; nor do I consider the post mortem appear- ances sufficiently characteristic to warrant the infer- ence that these animals died of a fever identical with the malarial fevers to which the human race INTERMITTENT FEVER. 325 is so subject; especially in view of the fact, that infection did not occur in the natural way, that the rabbit is very subject to various forms of septi- caemia, and that prior to these experiments no eVi- dence had ever been presented to show that the rabbit experiences any harm from respiring an atmosphere charged with malaria. Professor Ceri, however, claims to have produced in rabbits intense febrile paroxysms of a decidedly intermittent type, and continuing for a long period, by the hypodermic injection of artificially cultured malarial soil exposed for ten days to a temperature of 35° to 40° C. This is a very definite statement, and, if sup- ported by temperature-charts showing the fact, would have great weight. In a recent report (March 18, 1883) to the Italian Minister of Agriculture, Tommasi-Crudeli refers to the production of intermittent (?) fevers in the lower animals by the subcutaneous injection of the blood of malarial-fever patients, and states that he made extensive preparations to continue his experiments in this direction during the year 1882 ; but he was unable to carry out his inten- tion for the reason that not a single case of perni- cious fever was received during that period into the Roman hospitals.1 Here, then, we have a confession which makes it evident that the pernicious fever, ascribed to malaria, 1 Quoted from a paper in the Med. Record of August 18, 1883, by Dr. C. P. Russell. 326 BACTERIA IN INFECTIOUS DISEASES. by the author referred to, differs from ordinary malarial fevers — intermittents and remittents — which also prevail in Italy, in the essential par- ticular that it is an infectious disease, and may be transmitted to the lower animals ; as well as in the fact that it is a continued rather than a paroxysmal fever. The writer has long suspected that the continued pernicious fevers of the Roman Campagna, and of other parts of Italy, differ essentially from the or- dinary intermittents and remittents of this country, and that, while there is undoubtedly a malarial element, in a certain proportion of the cases at least, there is another etiological factor to which the continued and pernicious form of development manifested by the morbid phenomena must be ascribed. We know that malaria may be associ- ated with the specific poisons of typhoid and of yellow fevers in such a way as to produce atypical forms of these diseases, and it seems not impro- bable that the Roman fever is in truth one of these mixed or hybrid forms of disease. In this case the bacillus of Klebs and Tommasi-Crudeli, if it has any etiological import, is probably the factor to which the continued and pernicious form of this fever must be ascribed, rather than the malarial germ, which the authors named had undertaken to discover. Professor Ceri's experiments relating to the germicide power of quinine are extremely impor- tant and interesting. But it is well to remember INTERMITTENT FEVER. 327 that, if a dose of ten grains passed at once into the blood of an adult weighing one hundred and sixty pounds, the proportion which it would bear to the whole mass of blood in the body (estimated at twenty pounds) would be only 1 : 11,520; whereas Professor Ceri's experiments lead him to the con- clusion, " that the muriate of quinine in the pro- portion of 1 : 800 prevents the development of any infectious germs." 1 The preventive power for the Bacillus malarice, however, was found to be greater than this, and in a series of eighteen experiments in which culture- solutions were infected " with a drop of blood [con- taining the Bacillus malarice] of a rabbit into which had been injected cultures of malarial soil, the development continued absent up to 1 : 2,000, and at 1 : 2,250 it was aseptic. The Bacillus malarias did not develop in the fertile cultures, which con- tained only vibriones." The writer is not disposed to underestimate the value of these researches, but in a spirit of scien- tific conservatism would remark as follows : — First. — Fowler's solution of arsenic also cures intermittent fever; and the germicide power of this remedy is practically nil, as determined by the writer in a series of experiments in which the micrococcus of pus served as a test-organism. In the proportion of forty per cent it failed to kill this organism. Its power of restricting the devel- 1 Quoted from translation by Hugo Engel in " The Medical Times/' Philadelphia, Dec. 10, 1882, p. 198. 328 BACTERIA IN INFECTIOUS DISEASES. opment of the Bacillus malanw should be tested as a check on the conclusions which may be too hastily drawn from Professor Ceri's experiments with quinine. Second. — It is not impossible that the pernicious malarial fevers of Italy may differ essentially from the ordinary remittents and intermittents of this country, and that their continued form is due to a septic complication, which may result from invasion of the blood by a pathogenic organism peculiar to that country or to the tropical and semi-tropical regions where pernicious fevers are most prevalent. Third. — Fat-granules are found in the white corpuscles of the blood of yellow fever, — which disease resembles the pernicious malarial fevers in many particulars, - — which bear so strong a resem- blance to the spores of bacilli that a mistake might easily be made.1 (See p. 425.) Several of the observers named found spores " included in the white blood-corpuscles, which were sometimes so numerous as to seem to fill them completely." Fourili. — No great significance can be attached to the finding of bacterial organisms post mortem in the blood and tissues, especially in warm cli- mates, unless the examination is made immediately after death. And even then we must admit the possibility that such organisms may migrate from the intestine, where they are always present in abundance, during the last hours of life, when the circulation is feeble, and the vital resistance of the i Compare Fig. 3, Plate XII. and Fig. 3, Plate VIII. INTERMITTENT FEVER. 329 cells intervening between the lumen of the intes- tine and of its capillary vessels is very feeble, or quite lost. Finally. — The writer's observations lead him to be suspicious as regards the pathogenic role of or- ganisms in the blood, which are few in number and require diligent search for their demonstration. And the possibilities of accidental contamination are so great, when a drop of blood is drawn from the body of a patient with the greatest possible precautions, that the finding of a rod or of a sphere supposed to be a bacillus or a micrococcus requires verification by the finding of at least several more rods or spheres of the same kind in the same specimen ; and by the use of staining reagents and the test of cultivation. A more recent discovery than the Bacillus mala- rice of Klebs and Tommasi-Crudeli is the Oscillaria malarice of Laveran (1881). This discovery is also confirmed by Richard. The first-named author says : — " There exist in the blood of patients attacked with malarial fever pigmented parasitic elements, which pre- sent themselves under three principal aspects. ... " The parasitic elements are only found in the blood of patients sick with malarial fever, and they disappear when quinine is administered. "They are of the same nature as the pigmented bodies which exist in great numbers in the vessels and organs of patients dead with pernicious fever, and which have heretofore been described as melanotic leuco- cytes." 330 BACTERIA IN INFECTIOUS DISEASES. These parasites are described as being some- what smaller than the leucocytes of the blood, as sometimes resting and sometimes exhibiting amoeboid movements, and as sometimes having three or four long, motile filaments attached, which are very difficult to see except when they are in motion. They contain pigment granules, commonly arranged in a circle. Richard states that the malarial parasite of Laveran invades the red blood-corpuscles, where it is first seen as a minute round spot upon the circumference. In other corpuscles it is larger, and about the margin is seen a circle of black nodules. In others still the parasite has reached such a size that only a narrow, transparent zone remains between its circumference and the cell-wall of the red corpus- cle, and no trace of the haemoglobin remains. The oscillaria is, however, still surrounded by a ring of black nodules. The parasite now escapes into the blood-serum. The motile filaments de- scribed by Laveran are also referred to by Rich- ard, who states that in some cases they alone perforate the cell-wall of the red corpuscle, which is moved about in a peculiar manner by their oscillations. The presence of these parasites was demon- strated in the blood of every case of malarial fever observed by Richard, and frequently they were very numerous. We shall not attempt to estimate the scientific value of these observations, but would remark LEPROSY. 331 that Laveran and Richard, in their researches, seem not to have encountered the Bacillus malarice, although the announcement of its discovery had been made two years prior to the date of their investigations, and should have prepared them to find it if present in the blood of malarial cases studied by them. LEPROSY. — In 1879 Hansen, in a report to the Medical Society of Christiania (Norway), stated that he had " often, indeed generally, found, when seeking for them in leprous tubercles, small, rod- shaped bodies in the cells of the swelling." These rods were not found, however, in blood recently taken from leprous patients. Certain brown cells were also described in this report as peculiar to leprosy. In a later communication (1880) Han- sen says : — " I have by this preparation [staining with methyl- violet] obtained confirmation of my earlier supposi- tion, that the large brown bodies, after all, are nothing else than either masses of zoogloea, or collections of bacilli which are enclosed in cells. By looking at Fig. 4 [Fig. 16], which represents tumor-cells treated with osmic acid, drawn from preparations made in 1873, one is easily able to form an idea how these same cells, by a constantly increasing number of small rods, at last become quite overloaded, and thus obtain the appearance of being filled with fine granules, since the single rods cannot then be distinguished. . . . " Since writing the above I have also been so fortu- nate as to obtain bacilli, finely colored, in a section of a tubercle hardened in absolute alcohol. . Bacilli 332 BACTERIA IN INFECTIOUS DISEASES. Fig. 16. * Cells from leprous tubercle containing the Bacillus lepree. Copied from plate illus- trating Hanson's paper in Quart. J. Micr. Sci., Jan. 1880. are found in all parts of the section, either singly, or more frequently in groups, fully corresponding to those occurring in the cells. I furnish a drawing of two groups taken with Zeiss's immersion system -^ and eye- piece No. 4." (See Fig. 17.) One observer, Kober, claims' to have found the bacillus of Hansen in the blood of leprous pa- tients ; while Edlund ascribes the disease to a micrococcus which he finds in the blood, as well as in the leprous tubercles. Neisser, also, says that micrococci are always present in the epider- mis, although he confirms Hansen as to the pres- LEPEOSY. 333 / ence of bacilli in the leprous tubercles, and also in the liver, spleen, testicles, lymph-glands, and other parts. ( Query : How much time had elapsed between the death of the patients and the autopsies.) According to Neisser : — "The bacilli have the form of small, slender rods, with a length about half the diameter of a red blood-corpuscle, and about four times as long as broad. They approach most nearly the bacilli connected with the septicaemia of the mouse, but are not so fine." [According to Koch, they very Fig. 17. Closely resemble the bacillus Of Copied from Hansen's paper above tuberculosis.] " They are in- visible in uncolored sections, but beautifully seen when tinctured with fuchsin and gentian-violet. Their rela- tive position and distribution vary greatly, according to the part where they are found. They lie either two or three behind one another, apparently forming a long, sometimes curved, thread ; or six or seven lie parallel to one another ; or large numbers are associated in all directions into a confused mass, which is only with diffi- culty resolved into its elements. At a later stage of the leprosy the rods break up into granules ; but whether these are the result of disintegration, or must be regarded as spores, is doubtful. The bacilli were found in greatest quantities in the skin ; next to that, in the testicles ; also in the spleen and liver ; they were not found in the marginal parts of the lymph- canals ; the kidneys were free from them." l 1 Quoted from Journal of the Royal Micr. Society, Ser. II. Vol. I. Part 2, December, 1881, p. 928. 334 BACTERIA IN INFECTIOUS DISEASES. The presence of these bacilli in leprous tuber- cles, etc., has been confirmed by several observers in addition to those mentioned. Recently Dr. Thin, an experienced microscopist and mycolo- gist, has reported that he finds, in the skin of Chinese lepers, a bacillus of the size and form, and same staining qualities, as that described by Hansen.1 It is said that the bacillus is not found in the anaesthetic form of the disease. The writer examined the blood of lepers in the Charity Hospital, New Orleans, during the sumT mer of 1880, with a negative result, so far as the direct examination was concerned. But in cul- ture-cells in which a drop of blood, protected from the external air, was supplied with oxygen from a small air-space, hermetically enclosed, micrococci developed, which may be seen in the heliotype re- production of a photo-micrograph made from such a specimen, Plate II. Fig. 2. Inasmuch as these lepers had upon the face and hands ulcerated tubercles, the pus from which was doubtless infested with micrococci, very little importance was attached to the fact that micro- cocci made their appearance in these culture-cells. For the chances of accidental contamination, of a drop of blood drawn from the finger, by micro- cocci from the surface of the body, were so great as to give but little value to the culture-experi- ment, notwithstanding the fact that the precaution was taken to wash the finger with alcohol before 1 British Medical Journal, Aug. 5, 1882, p. 231. LEPROSY. 335 making the puncture. The writer's own experi- ments have since shown that this precaution is probably inadequate ; for the micrococcus of pus is not killed by exposure for two hours to 25 per cent alcohol. Up to the present time, the supposition that the bacillus of Hansen bears a causal relation to lep- rosy depends for its support entirely upon the fact th#t it is found in the leprous tubercles, etc. It is not well established that these bacilli have dis- tinctive morphological characters and staining re- actions. Indeed, Koch finds that they closely resemble his bacillus of tuberculosis in both these particulars. But even if this bacillus were proved to be peculiar to leprosy, in the absence of suc- cessful inoculations with pure cultures its causal relation to the disease must remain ^in question ; for, in view of what we know of the habits of the bacteria generally, there is* nothing improbable in the supposition that this particular species is able to invade tissues of a low grade of vitality, and finds in the leprous tubercles the pabulum necessary for its development. If, however, lep- rosy is truly an infectious disease, which seems to be a matter of considerable doubt, the rapidly accumulating evidence in favor of the parasitic- germ theory, in explanation of the etiology of these diseases, lends strong probability to the first-mentioned hypothesis. Hansen has endeavored to inoculate rabbits with leprosy, by introducing portions of the leprous 336 BACTERIA IN INFECTIOUS DISEASES. growths, especially the tubercles, under the skin of these animals. He says, " I was not lucky in any of these attempts." (1880.) More recently he has inoculated a monkey, which, at the date of his report, had been under observation for six months, without having developed any symptoms of the disease. But as the time of incubation in man is said to be a year or more, this experiment is not considered decisive. The bacilli have been successfully cultivated by their discoverer upon gelatinized blood-serum. In these cultures development commenced after an interval of three or four days, and the bacilli often presented nodular enlargements at the ex- tremities, which were believed to be due to the formation of spores. In these cultures filaments formed, macje up of a number of bacilli, and these were often so abundant as to form an entangled • net-work. The fact that these bacilli multiply and develop spores in a culture-solution, within a few days, while the period of incubation in leprosy is "at least a year," seems a little difficult to recon- cile with the supposed etiological role of these parasites. MALIGNANT (EDEMA. — According to Koch, a frequent source of error in experiments on anthrax arises from accidental contamination of the culture- fluids by a bacillus which closely resembles B. au- tJmwi*. This organism is called the bacillus of malignant oedema, and the disease to which it gives MALIGNANT (EDEMA. 337 rise has been especially studied by Gaffky, who states that the organism is apparently identical with the vibrion septuple of Pasteur. Although very similar to the anthrax bacillus, Koch points out certain morphological characters which distinguish the one from the other. The anthrax bacilli are a little broader than the others, and the joints have concave extremities ; whereas the others are rounded at the extremity. B. an- thracis is motionless, while that of malignant oedema is usually in active motion. According to Ewart, the anthrax bacillus, also, is motile during certain stages in its life-history : — " The disease is readily produced by the introduction of a small quantity of garden-earth under the skin of an animal (rabbit, guinea-pig, or mouse). The animals become ill very soon, there being no distinct incubation period, and death occurs after twenty-four to forty-eight hours. Spreading from the point of infection, the sub- cutaneous cellular tissue and the intermuscular cellular tissue become oedematous and reddened, the spleen is enlarged, soft, and of a dark reddish-blue color ; but the other organs are not altered to the naked eye. No bacilli, or only very few, are found in the blood of the heart immediately after death ; but the fluid obtained after section of the various organs contains numbers of these moving rods. The longer the time which has elapsed after death, the more numerous do the bacilli in the tissues and blood become. They grow best in the dead body, thus differing from other pathogenic organ- isms. On section of the organs, the bacilli are found in the cellular tissue, almost exclusively towards the sur- 22 338 BACTERIA IN INFECTIOUS DISEASES. face ; they apparently spread into the organs from the cellular tissue around. They may also form plugs in the capillaries, though this is rare. In some cases putrefaction occurs rapidly, but in others it is apparently retarded. " With regard to the cultivation of these organisms outside the body, it has been found by Pasteur, Joubert, etc., that they will not develop in presence of oxygen, but readily grow when carbonic-acid gas is substituted for oxygen in the cultivating flasks. This observation is confirmed by Gaffky, who grew them in the interior of potatoes removed from the air. These bacilli caused death when injected into the subcutaneous cellular tissue, thus showing that they were the true materies morbi. " Of great interest is the question of the relation of these organisms to those found by Lewis in the blood of asphyxiated animals, especially of rats, — an observa- tion confirmed by Gaffky. These organisms are found most frequently in the blood of horses, and Koch ex- plains this by the slower cooling of their bodies. In smaller animals, these organisms, which probably come from the intestine, do not develop rapidly, unless the body be kept at a temperature of about 38° C. Dr. Gaffky asphyxiated a guinea-pig, and then placed the body in an incubator. In twenty-four hours the body was much swollen from gas-development ; and from the natural orifices bloody fluid exuded, containing numerous bacilli indistinguishable from oedema bacilli. Everywhere throughout the body, more especially in the subcutaneous cellular tissue, these bacilli were present in large numbers. A drop of fluid from the cellular tissue was injected into a second guinea-pig. This animal died on the following day, with the typical appearances of malignant oedema. A minute quantity MILK SICKNESS. 339 of the oedematous fluid, diluted with distilled water, and injected into a third guinea-pig, was followed by the same result."1 MILK SICKNESS. — This is an infectious disease which prevails in certain rural districts in the United States, and which is said gradually to recede before the advance of improved agricul- ture : — " In its source, in unimproved marshy localities, it closely resembles the malignant anthrax ; also, in its communicability to all animals ; but it differs essen- tially in that it fails to show local anthrax lesions, in place of which it expends its energy on the nerve centres, producing great hebetude and loss of muscular power. According to Dr. Phillips it is characterized by the presence in the blood of a microzyme (spirillum) like that seen in relapsing fever. The germ is probably derived from drinking-water, or the surfaces of veg- etables, as certain wells are found to infect with cer- tainty, and the disease has been repeatedly produced by feeding upon particular plants (Rims toxicodendron, etc.). That these plants, in themselves, are not the pathogenic elements, is shown by their innocuous prop- erties when grown in places out of the region of the milk-sickness infection. It seems altogether probable that here, as in malignant anthrax, we are dealing with a microzyme which has developed pathogenic properties, and which can be reproduced indefinitely in the bodies of living animals. The great danger of this affection consists in the conveyance of the germ with unimpaired potency through the flesh and milk, and through man- ufactured products of the latter, — butter and cheese. 1 Quoted from The British Medical Journal, July 15, 1882, p. 99. 340 BACTERIA IN INFECTIOUS DISEASES. Some even hold that in animals giving milk the system does not suffer materially, but that it is saved by the drainage of the germs through the mammary glands, and that thus a milk-sick cow may remain for a con- siderable time unsuspected. . . . The disorder proves fatal in man as in animals." l A careful study of this disease by the experi- mental method would probably demonstrate its parasitic nature ; and it is extremely desirable that its etiology may be worked out, both in the inter- est of science and of medicine. MEASLES. — Coze and Feltz state that bacteria are found in the blood of measles, of extreme minuteness and great mobility. In the period of invasion the nasal mucus contains small "bacteri- forin elements." The inoculation of this blood did not produce the death of rabbits ; but these animals were sick for two or three days, as the result of such inoculations, and " very slender and active rods " were found in their blood (Magnin). Klebs, also, found micrococci in the trachea, and in blood taken from the heart of infants that had fallen victims to this disease. In the blood, preserved in capillary tubes, these micrococci developed in spherical masses. Braidwood and Vacher describe certain small spherical bodies first found by them in the breath of children in the acute stage of the disease, which they believe to be the contagious elements. These 1 Prof. James Law, National Board of Health Bulletin, Vol. II. No. 4, p. 456. PLFURO-PNEUMONIA. 341 are "sparkling, colorless bodies, something like those found in vaccine, but larger." Some were spherical, others were elongated, with sharpened ends. The breath of healthy children did not contain these sparkling bodies. These bodies were also found in the lungs and liver of two children who died of measles. Keating has recently (1882) reported the find- ing of micrococci in the blood of malignant measles, and their absence in cases of mild type. He says : " The micrococcus is found in the con- tents of pustules and vesicles, and also in the blood taken from the measles-papule in mild cases, without its being present in the blood taken from the punctured finger. In severe cases, called malignant in this paper, owing to the rapid ap- pearance of morbid symptoms, the blood shows, early in the attack, numerous patches of rnicro- coccus in the field." These observations were verified by Formad. PLETJKOPKEUMO^IA. — The infectious disease of cattle known as pleuro-pneumonia has been studied experimentally by Willems, Banti, Bouley, Leblanc, Bruylants, Verriest, and others, and strong evidence has been adduced in favor of the view that it is due to a parasitic micro-organism. In 1852 Willems pointed out the existence of certain peculiar corpuscles in the lymph obtained by incision of the lung of an animal dead from this disease. This observation has been confirmed 342 BACTERIA IN INFECTIOUS DISEASES. by others, and Bruylants and Verriest describe the organism, which they were able to cultivate in sterilized fluids, as a micrococcus, sometimes isola- ted, sometimes in pairs, and sometimes in chains of 3-10 elements. The form is slightly oval, and the size varies considerably, the largest measuring 1 jji in diameter. Protective inoculations are successfully practised in this disease. INFECTIOUS PNEUMONIA. — That there is an in- fectious form of pneumonia in man is now pretty generally admitted upon clinical evidence ; and several observers have described micro-organisms supposed to bear a causal relation to this disease. Klebs claims to have produced lobular pneumonia in rabbits by injecting the sputum of patients suf- fering from pneumonia, in which he found an or- ganism called by him monas piibnonale. Friecllander, also, found micro-organisms in eight successive cases in the expectoration and in sections of pul- monary tissue. These micrococci wrere elliptical in shape, one micro-millimetre in length, and two-thirds //, iii^ breadth. They were usually in pairs, but also occurred in chains, and were found most abundantly in the fibrinous expectoration, and in grayish-red hepatization, The writer would call attention to the fact that these oval micrococci seem to resemble closely those found in the blood of a rabbit killed by the subcuta- neous injection of human saliva. (See Fig. 3, Plate PYAEMIA IN RABBITS. 343 / ' IX., and also p. 359.) Leyden has demonstrated the presence of numerous micrococci corresponding with those described by Friedlander in exudation fluid obtained during life from a patient suffering from severe croupous pneumonia. The fluid was withdrawn by means of a hypodermic syringe. Gunther. also, has obtained the same result from an exploratory puncture of hepatized lung. On the other hand, negative results were obtained by Leyden in two milder cases of pneumonia in which fluid was withdrawn from the inflamed lung; and in an epidemic described by Kiihn, search for micro-organisms gave a negative result. IN KABBITS, Koch. — After failing to produce a general infection in rabbits by the injection of putrid blood, Koch succeeded with a fluid obtained by macerating for two days in dis- tilled water a bit of the skin of a mouse. The animal died at the end of one hundred and five hours, and a purulent infiltration of the subcuta- neous cellular tissue was found, extending from the point of inoculation as far as the hip behind, and to the middle of the belly below. The peri- toneal cavity contained a turbid fluid, and its walls were covered in places by white patches. The liver was covered with a fibrinous exudation, and presented a grayish mottled appearance ; upon section it showed gray, wedge-shaped patches. In the lungs were found dark red patches, the size of a pea. 344 BACTERIA IN INFECTIOUS DISEASES. A syringeful of blood from this animal killed a second rabbit in forty hours. Rabbit No. 3 was killed in fifty-four hours by three drops of blood from No. 2 ; one drop from No. 3 killed No. 4 in ninety-two hours; one-tenth of a drop from No. 4 killed No. 5 in one hundred and twenty-five hours. The pathological appearances in this se- ries of rabbits were similar to those noted in the first, viz. : — " Local purulent cedematous infiltration of the sub- cutaneous cellular tissue ; metastatic deposits in the lungs and liver ; swelling of the spleen and peritonitis. These appearances harmonize so closely with those commonly designated as pyaemia that I do not hesitate to use that term for the disease under consideration. " On microscopic examination micrococci are found in great numbers everywhere throughout the body, and more especially in parts which have undergone altera- tions visible to the naked eye. These micrococci are, for the most part, single or in pairs, and their meas- urement is therefore difficult. Ten measurements of pairs of micrococci differed but little from each other, and gave .25 //,. as the average diameter of a single individual." It will be noticed that this is much less than the size of the oval micrococcus which produces septi- caemia in rabbits. " As regards size, therefore, they stand midway between the chain-like micrococcus of the progressive gangrene of the tissues and the zoogloea-forming micro- coccus of the cheesy abscesses of rabbits. Their relation to the blood-vessels can be best seen in the renal capil- PYAEMIA IN RABBITS. 345 Fig. 18. laries, and I have therefore selected a small vessel from the cortex of the kidney for delineation (Fig. 18) u In the interior of the vessel, at siys that it is more difficult to distinguish the individual elements in the chains than in the case of spherical micro- SYPHILIS. 381 (' cocci. These oval elements are found single, in pairs, or in chains of four or five, which greatly resemble long rods with rounded ends. This description agrees with that of Aufrecht: " For the demonstration of the organisms in recent preparations, Birch-Hirschfeld prefers potash, by the clearing action of which the inicrococci are visible in the tissue, 011 account of their strong refracting power. In a broad condylonia, they lie, for the most part, in small aggregations in the papillso, and in many of the cells of the adjacent layer of the rete Malpighii. They may be readily detected in the juice of a recently ex- cised condylonia, by tinting in the ordinary way ; and of the various staining agents, Birch-Hirschfeld con- cludes that fuchsin and gentian-violet are the best. In the growths in internal organs the smallest inicrococci are most abundant, and the larger forms seen in the cou- dylomata are seldom met with. In gurnmatous scars they are sought for in vain. In more recent gummatous products they were most abundant in parts which had the aspect of growing granulation tissue. They were partly scattered, partly aggregated into groups, which never exceeded a granulation-cell in size ; they were also distinctly seen within the cells. Many epithelioid cells seemed to have their nuclei filled with these or- ganisms." l Dr. Bermann of Baltimore finds in absolutely fresh specimens of indurated chancres, " collections of micrococci and fungoid growths, firmly adhering to and partly blocking the lumina of most of the lymphatic vessels." According to this observer, the 1 London Lancet, December 2, 1882. 382 BACTERIA IN INFECTIOUS DISEASES. micrococci of syphilis are small, strongly refract- ing bodies; resembling those described by Klebs. Recently Dr. Morison of Baltimore has made a careful study of the bacteria found in chancroids and in syphilitic lesions, in the wards of Professor Neumann of Vienna. As he resorted to the most approved methods of staining, and seems to have Fig. 20. Hard chancre secretion with bacteria, magnified 850 diameters. (Drawn by Heitzmann. ) exercised special care in collecting and mounting his material for microscopic examination, his obser- vations are of value, and I have taken the liberty of copying his figures from the " Maryland Medical Journal" of January 1, 1883, in which his paper was published. (See Figs. 20 and 21.) No satisfactory proof has yet been offered in support of the view that airy one of the organisms above described is the veritable germ of syphilis ; SYPHILIS. 383 and it is evident that the greatest caution must be exercised in drawing any conclusions as to their etiological import. For there is nothing improbable in the supposition that tissues of a low grade of vitality may be invaded by parasites which have no causal relation to the morbid process; and in view of what we know of the extended distribu- Fig. 21. Soft chancre secretion with bacteria, magnified 850 diameters. (Drawn by Heitzmann.) tion and infinite variety of organisms of this class, their absence from the secretions of an open ulcer would be more remarkable than their presence. In a second communication, dated March 23, Dr. Morison states that a modification of his method of staining has enabled him to demonstrate that the rods seen in Fig. 20 are really formed of closely united cocci, corresponding with those de- scribed by Birch-Hirschfeld. He further says : — 384 BACTERIA IN INFECTIOUS DISEASES. " The result of these recent experiments is such that I am not only forced to deny the pathogenic nature of the micro-organisms described in rny first communication, but also to add that I am convinced their presence in the secretions was due to external influences." Klebs claims to have produced syphilis in the monkey, and Martineau and Hamoine to have communicated the disease to young pigs (Morison). But with these exceptions, so far as the writer is aware, attempts to inoculate syphilis in the lower animals have given negative results. TUBERCULOSIS. — The experimental researches of Villeman, Tappeiner, Colmheim, Toussaint, and others, having apparently established the fact that tuberculosis is an infectious disease, the medical profession was not unprepared for the discovery, first announced by Koch in the spring of 1882, of a parasitic micro-organism in tuberculous material, bearing a causal relation to the disease in question. Coming from Koch this announcement had great weight and at once received the most attentive consideration in all parts of the civilized world ; for he was already well known to be both a skilful and a cautious investigator. The experimental proof offered in favor of the view that the bacillus discovered in the sputum of tuberculous patients, and in recent tubercles in the lungs and elsewhere, was the veritable cause of tuberculosis, seemed so convincing, that it might have been received almost without question, but for the fact that other experimenters had pre- TUBERCULOSIS. 385 viously found that tuberculosis in animals may result from inoculation with a variety of organic products of non- tubercular origin, and even from the inhalation of inorganic particles ; which also is recognized as a cause of pulmonary consumption in man. As an example of the numerous experi- ments of this kind, we may refer to the results obtained by Brunet, who inoculated seven rabbits with cancer, six with simple pus, and six with tuberculous material. Of those, fourteen became tuberculous, namely, six of those inoculated with cancer, three of those inoculated with pus, and five of those inoculated with tuberculous matter. Schottelius found that miliary nodules in the lungs resulted, in dogs, alike from inhalation of pulverized — spray — sputum of bronchitis and of phthisis. Toussaint affirms that the tubercular deposits resulting from inoculation with non-tubercular material are not infectious, and that experimental pseudo-tuberculosis may be distinguished from tuberculosis proper by inoculation experiments, although the pathological anatomy of the two diseases is identical. Koch, on the other hand, does not admit that tuberculosis can be produced by material from which living tubercle bacilli or their spores are un- questionably excluded. In his own experiments he found that in all cases where the material used for inoculation contained living bacilli or spores, the result was positive in animals liable to infec- 25 386 BACTERIA IN INFECTIOUS DISEASES. tion ; while when the material inoculated did not contain these bacilli or their spores, a negative result was obtained. Thus, in several cases, ex- periments were performed with the contents of a scrofulous gland and with various other material proved by examination to be free from bacilli, and in no instance did tuberculosis follow. The posi- tive results obtained by other experimenters with non-tuberculous material are explained by the sup- position that tubercle bacilli or their spores have been introduced at the same time. It is evident that this accidental inoculation would be very apt to occur in laboratories where tuberculous animals had been kept under observation, and especially where proper precautions are not taken as regards cleanliness of the cages in which animals are kept, and the isolation of those which are subjected to inoculation experiments. According to Koch, the tubercle bacillus is a slender rod from a quarter to a half of the diameter of a blood-corpuscle in length, and presents certain distinctive characters as regards its behavior with staining reagents. The various methods of stain- ing this bacillus are given in 'PART THIRD of the present volume. The bacilli are found in consider- able numbers in tubercles of recent formation, more especially at the border of the cheesy masses. They are abundant in the giant-cells, and seem to possess a special relation to these cells. They are not so abundant in old tubercles, although they are seldom entirely absent. By placing a small por- TUBERCULOSIS. 387 tion of a recent tubercle in blood-serum or distilled water, they may be recognized with a suitable objective and illuminating apparatus, without the use of staining reagents. An examination made under these circumstances shows that the bacilli are motionless, and in some rods spores of oval form may be distinguished. At the time of his first report, Koch had examined in man, " Eleven cases of miliary tuberculosis, twelve cases of cheesy broncho-pneumonia, one case of tubercle in the brain, and two cases of intestinal tuberculosis." In all of these the bacilli were present. They were also found in freshly extirpated scrofulous glands. Among the lower animals they were found in ten cases of perlsuckt, in three cases of so-called bron- chiectasis in cattle ; in three monkeys, nine guinea- pigs, and seven rabbits, which had spontaneous tuberculosis ; and in one hundred and seventy-two guinea-pigs, thirty-two rabbits, and five cats, which had been inoculated with tuberculous material, or with pure cultures of the bacillus. The gelatine culture-medium which had been previously recommended by Koch was found not to be suitable for the cultivation of the tubercle bacillus, as the advantage of solidity is lost when this is heated to 98° Fahr. Jellified blood-serum, prepared as directed on page 163, was found, how- ever, to fulfil all the required conditions, and was used by Koch in his culture experiments. Portions of tubercles removed, with proper precautions to prevent contamination, from the bodies of persons 388 BACTEEIA IN INFECTIOUS DISEASES. recently dead of tuberculosis, or from the lower animals, victims of spontaneous or of induced tu- berculosis, were placed upon the surface of the sterilized blood-serum, and the vessel containing it was kept in a culture-oven maintained at a tem- perature of 40° C. (104° Fahr.). During the first week no marked alteration occurred, unless other bacteria had gained access to the culture-medium, in which case the experiment was a failure. About the tenth day small points and scales became evi- dent, which slowly spread, and upon microscopical examination proved to consist of tubercle-bacilli. After fourteen days these bacilli were used to start* a new culture. This was accomplished by break- ing up the scales and transferring a minute quan- tity to the surface of culture No. 2. After transferring the bacilli in this way to several successive flasks, it was assumed that the origi- nal material was excluded, and that a pure cul- ture had been obtained. Inoculation of guinea-pigs with these pure cultures gave rise to tuberculosis with as great certainty as in those experiments in which tubercular material was used. In one ex- periment six newly bought guinea-pigs were ob- tained. Two of these were kept as lemoins, and the other four were inoculated with cultivated bacilli obtained in the first instance from the lung of a human being who had died of miliary tuber- culosis. In this instance five successive cultures had been carried out, the time required being fifty- four days. One of the inoculated guinea-pigs died TUBERCULOSIS. 389 on the thirty-second day, and all the rest were killed on the thirty-fifth day. All had extensive tuberculosis, and the Bacillus tuberculosis was found in the tubercles of the lungs, and of various organs. The two guinea-pigs not inoculated re- mained healthy. In another experiment four rabbits were taken. Into the eye of one pure blood-serum was injected ; the point of a syringe containing tubercle bacilli in blood-serum was introduced into the eye of a sec- ond. These were from a series of cultures carried out for 132 days. In this case the piston was not moved ; but the same material was injected into the eye of rabbit No. 3, and of rabbit No. 4. The animals were killed on the thirtieth day, and the following result noted : Rabbit No. 1 remained healthy ; rabbit No. 2 had typical tuberculosis of the iris, and the nearest lymphatic glands were swollen and infiltrated with yellowish nodules ; but the lungs and other organs were free from tubercles. Rabbits Nos. 3 and 4 had iritis and tuberculosis of the lungs. The presence of Koch's bacilli in tuberculous sputum has now been confirmed by numerous ob- servers in various parts of the world ; and the comparatively few failures to find the bacillus which have been reported by expert manipulators since the method of Ehrlich was published, are easily accounted for in other ways than upon the supposition that cases of tuberculosis occur in which no bacilli are found. Nevertheless we must 390 BACTERIA IN INFECTIOUS DISEASES. admit that there are cases, recognized by expert pathologists as undoubtedly tubercular, in which no bacilli can be found in tubercles obtained from the lungs post mortem. Thus Prudden, of New York, while recording the fact that he has, in a considerable number of cases of acute and chronic phthisis, found, almost invariably, the bacillus of Koch " in and about all of the cavities, in many of the larger areas of coagulative necrosis, and in a considerable proportion of the miliary tuber- cles;" yet reports two cases which form an ex- ception to this rule. In one, an abundance of miliary tubercles covered the lateral surfaces of both lobes of the left lung ; " most of these were of the usual giant-celled and epithelioid-celled type, with a more or less well-marked reticulum. In none of those examined was there well-marked cheesy degeneration. Six hundred and ninety-five sections, about .01 millimeter in thickness, were made from ninety-nine different tubercles from various parts of the tuberculous membrane, and stained in the usual manner by Ehrlich's method in several different lots. In not one of these six hundred and ninety-five sections could a single tubercle bacillus be detected, although all were examined with the most scrupulous care." In another case, "nine hundred and nine sections from a large number of peritoneal tubercles, from different parts of the affected surfaces, stained by Ehrlich's method, revealed, under the most search- ing scrutiny, no tubercle bacilli." In the same TUBERCULOSIS. 391 case, however, nodules at the apex of the lung, and the wall of a small cavity formed of shreds of necrotic tissue, of dense cheesy material, and in the outermost layers of tubercle tissue and ordi- nary dense connective tissue, proved to contain the bacillus in abundance in the walls and edges of the cavity i and in a few of the dense areas of coagu- lation necrosis in its immediate vicinity. But in the diffuse tubercle tissue, in the zones of simple pneumonia around the nodules, in the scattered fibrous tubercles in the lung and pleura, and in the well-formed tubercles in the bronchial glands, no bacilli could be found. Koch has received ample confirmation as to the presence of the bacillus described by him, in phthisical sputum • and its absence from the spu- tum of patients suffering from other diseases seems to be pretty well established, although Spina of Vienna claims that other bacteria behave pre- cisely towards staining agents as do the bacilli of Koch ; and, consequently, that the color-test can- not be relied upon for distinguishing this bacillus from the ordinary bacteria of putrefaction. The writer's observations are entirely in favor of the statement of the discoverer of the tubercle bacilli as to their peculiar color reaction when treated by Ehrlich's method; but, like many others, he has not been successful in demonstrating them by the method first proposed by Koch. A recent writer1 has collected the statistics, as 1 Dr. Ferguson, of Canada. See Med. Record, New York, July 21, 1883, p. 77. 392 BACTERIA IN INFECTIOUS DISEASES. published in various journals, and states that in 2,509 cases reported, the bacilli were found in 2,417- Koch, himself, recognizes, however, that this kind of evidence cannot be taken as proof of the causal relation of the bacillus to the morbid process which results in the formation of tubercles in various parts of the body. For it may be that the bacillus is present in tuberculous material simply because this furnishes the pabulum neces- sary for its development, and is absent from the sputum of bronchitis, for example, because this does not constitute a suitable culture-medium ; or because, being secreted from the surface of an inflamed mucous membrane, and quickly removed by expectoration, there is no time for the develop- ment of this bacillus, which Koch has shown re- quires at least a week before any evidence of multiplication is seen upon the surface of sterilized blood-serum. This time would, however, be af- forded in the cheesy contents of a tubercular nodule, or in a cavity where necrotic products were retained for a considerable time. A recent French writer, Cochez, claims that the sputum of phthisical patients constitutes a favora- ble culture-medium for the tubercle bacillus. The writer, also, has been inclined to believe that the bacilli are more numerous in sputum which has been kept for a day or two than in the same material when first obtained. This, if true, is not very favorable to the view that they are the cause of the morbid process which results in the forma- TUBERCULOSIS. 393 tion of miliary tubercles, although by no means directly opposed to this belief. An interesting communication relating to the finding of Koch's bacillus in pathological speci- mens which have undergone putrefaction, or in those which have been kept for some time in pre- servative solutions, has recently been made by Vig- nal. This author finds that " putrefaction, even very much advanced, does not seem to interfere with finding the tubercle bacillus. They are also found as easily in pieces kept a long time in 90 per cent and absolute alcohol, and in Muller's fluid, as in recent preparations/7 The morphological characters of the tubercle bacillus, as found in sputum, are delineated in Fig. 22. The bacilli are found both within and without the pus-cells, and seem to be espe- cially numerous in the epithelioid cells. They vary greatly in length, and are not infrequently curved or bent at an angle more or less acute. Not infrequently they . OCCUl' 111 pairS, Or in Vj.,1 i • little groups, and in some cases it is apparent that they contain endo- genous spores, or that they are made up of a chain of oval elements. . In my " Photo-Micrographs," Fig. 22. Koch's Bacillus tuberculosis, in sputum ; in stained by Ehrlich's method. X 1000 (G. M. S., del.). 394 BACTERIA IN INFECTIOUS DISEASES. Figs. 3 and 6, Plate XI., the bacillus is seen very indistinctly; but a close inspection of these figures will show a single bacillus containing spores in Fig. 6 ; and in Fig. 3 groups of two contained in an epithelioid cell, the outlines of which can barely be distinguished. Under the microscope these bacilli, which had been stained with fuchsih by Ehrlich's method, were beautifully shown as bright-colored rods, which contrasted strongly in appearance with the decolorized micrococci and putrefactive bacteria in the same specimen. But after a considerable number of trials, no better photographic result could be obtained than that here seen. The fact that the tubercle bacillus has not inva- riably been found by competent observers in recent tubercles, and, also, that numerous investigators claim to have produced tuberculosis in susceptible animals by inoculating them with non-tubercular material, must make us unusually exacting as re- gards the experimental evidence furnished by inoculations with pure cultures. The writer has already indicated his reasons for thinking that a series of cultures made upon the surface of a solid culture-medium is less satisfactory, as regards the exclusion of the original material, than where a fluid culture-medium is employed, which is per- vaded throughout by the organism under cultiva- tion. (See p. 311.) The force of this criticism will be more fully appreciated when it is remembered that Koch TUBERCULOSIS. 395 several bits of a recent tubercle to start a culture upon the surface of jellied blood-serum. It cer- tainly seems that, under these circumstances, four or five successive cultures are not sufficient to make it entirely certain that we have obtained a pure culture of the bacillus; and we may fairly demand that the culture experiment should be carried much beyond this point. This requires, however, an amount of skilful manipulation, and of patient waiting which it will be hard to obtain outside of Koch's laboratory; and especially in this country, where no government aid is extended to those who engage in investigations of this char- acter. When stimulated by the expectation of making a new discovery which will make his name famous, a man may accomplish wonders in explor- ing difficult and unknown territory. But when the ground is to be re-surveyed, and carefully mapped, it is not to be expected that individual enterprise will be equal to the undertaking. The writer speaks with feeling in this matter, for he realizes how imperfect his own attempt to repeat Koch's experiments has been. A summary statement of the results obtained in this attempt is given below, as originally reported in " The Medical News " of December 30, 1882 : — " If I have not been able to answer the question, Is tuberculosis a parasitic disease? the experimental data thus far furnished will at least be of use to the jury — the medical profession — in making up a final verdict ; which, I submit, would in the present stage of the in- 396 BACTERIA IN INFECTIOUS DISEASES. vestigation be premature. I therefore beg leave to retire from the witness-stand, and to take my seat with the jury, with the understanding that I may be recalled if circumstances enable me to add to the testimony already given. First, however, it may be well to make a summary statement of the evidence presented; which I do as follows : — " (a) The bacillus described by Koch is found in the sputum of phthisical patients in San Francisco. " (&) That this bacillus differs from the micro-organ- isms found in normal human saliva and in bronchi tic sputum, is proved by the color-test (Ehrlich's) and by culture-experiments (see below, i and j). " () negatively, by the favorable results of antiseptic treatment ; and the fact that when the access of micro-organisms is prevented by the integrity of the cutis, very severe lesions, attended with an abundant exudation of bloody serum, are com- monly recovered from without suppuration or any evil result from the resorption of this fluid and of inflammatory exudates. But this same material quickly attains poisonous properties in the presence of bacteria, and not only exercises a deleterious local effect, unfavorable to the repair of the injury, but its absorption now is attended with the most serious consequences. These facts, which are so generally recognized that it is unnecessary to present evidence in their BACTERIA IN SURGICAL LESIONS. 443 support, are in accord with the following propo- sitions which have been established by experimental research and may be accepted as fundamental truths upon which to base our reasoning as regards the role of the bacteria in surgical lesions. (a) The blood and tissues of healthy persons do not, under ordinary circumstances, contain bac- terial organisms. (£) Putrefactive decomposition of organic fluids is due to bacterial organisms. (c) Albuminous fluids, — e. g., blood and pus, which have undergone putrefaction, contain a potent poison, or poisons, which, in comparatively small amount, may produce death in the lower animals. We have here a sufficient foundation for the antiseptic treatment of wounds. But in addition to this there are .strong reasons for believing that certain species of bacteria have also the power of invading the tissues, and producing local necro- sis, when for any reason the vital resistance of these tissues is reduced, — e. g., from hemorrhage, from starvation, from crowd poisoning, from septic poisoning. Or the same result may perhaps occur when the vital resistance of the tissues is not below par, in consequence of the unusual vigor of the micro-organisms, developed as a result of unusually favorable conditions of environment. As, for ex- ample, when a healthy man, recently wounded, falls a victim to hospital gangrene as the result of infection in a crowded ward, in which this in- 444 BACTERIA IN SURGICAL LESIONS. Fig. 28. fectious disease was in the first instance developed de novo. The purulent discharge from wounds not treated antiseptically always contains micro-organisms. These are mainly micro- cocci and short rods like those shown in Figs. 28 and 29, which are copied from Cheyne's recent work on "Antiseptic Surgery." The micrococci repre- sented in Fig. 28 were ob- tained by cultivation in cu- cumber infusion, from a Micrococci from a wound treated -t •* y • n asepticaiiy, growing in infusion wound treated asepl .cally. SSfciTwS The organisms represented in Fig. 29 are from a case of compound dislocation of the thumb not treated aseptically. The rod-bacteria in this figure are doubtless septic bacteria, properly so called, which give rise to the putrefactive decomposition of albu- minous fluids. The observations of Cheyne show that these may be excluded from the secretions of wounds by antiseptic treatment, and that, in this case, the pus discharged from such wounds pre- sents no evidence of putrefaction, although, in certain cases, micrococci are found in this pus formed beneath antiseptic dressings. This is ex- plained by the greater resisting power of micro- cocci to antiseptic agents. Cheyne says : 44 Micrococci prefer acid fluids ; most bacteria prefer alkaline or neutral fluids. BACTERIA IN SURGICAL LESIONS. 445 " Micrococci grow, readily, in fluids containing pro- portions of carbolic acid in which bacteria only grow with difficulty" (1. c., p. 244). The experiments of the writer have not shown any difference as regards the action of carbolic acid in preventing the develop- ment of these different or- ganisms in culture-fluids ; but in the case of boric acid and Of sodium biborate a very marked difference was observed, the micrococcus of pus developing freely in the presence of 0.25 per cent of boric acid, while B. termo failed to develop in the presence of one-half this amount. It is pro- bable that free access of oxygen in the culture- experiments, and its exclusion, to some extent at least, from the surface of wounds treated anti- septically by Lister's method, is an advantage in favor of the micrococcus in the latter case ; for we know that this may multiply freely in the absence of oxygen in the pus of a closed abscess. While there is no question as to the injurious effects of putrefactive bacteria in the discharges from wounds when these are retained upon an absorbent surface, or in a sinus or pus-cavity, the role of the micrococcus of pus has not been so well established. . According to one view, inflamma- Fig. 29. Specimen of discharge taken from a case of compound dislocation of the thumb not treated asepti- cally. X 1450. (From Cheyne's "Antiseptic Surgery.") 446 BACTERIA IN SURGICAL LESIONS. tion results in the formation of pus only when this micrococcus is present, and because of its presence. On the other hand, it has been claimed that it is simply present because it finds in pus a suitable culture-medium, and that its presence in this fluid is without significance. Cheyne is in- clined to look upon this micrococcus as compara- tively harmless ; and without doubt it may be present in the pus secreted by wounds which are healing in a most satisfactory manner. Cheyne says : "It is certain that they do not cause putrefaction, but they always cause a sort of sour, sweaty smell in fluids, — a smell which can be recognized in whatever fluid they grow : in other words, they are associated with a peculiar fermentation. Now, the products of this fermentation are but little irritating. They have no acrid taste, nor do they feel pungent when applied to a cut surface. Hence, probably, it is that we find wounds in which these organisms exist, even in large numbers, appear often unaffected by their presence. " Nevertheless, they can hardly, under any circum- stances, be indifferent, and I think I have observed that in .some cases, after they have got in, the wounds do not behave quite as typically as usual ; i. e., there may be a trace of suppuration, or a sinus takes longer to heal than one had any reason to expect." To test the possible local pathogenic action of the micrococcus of pus the writer made the follow- ing experiment : " Exp. No. 12. — August 4. — An incised wound was made with scissors, removing a fragment of skin upon BACTERIA IN SURGICAL LESIONS. 447 each thigh of a half-grown rabbit. The wound upon the right thigh was moistened with a culture-fluid (twentieth culture) containing the micrococcus from gonorrhceal pus. The wounds were then dressed with dry tow, and a bandage applied. Both healed kindly without any undue inflammation, and no difference was observed between the two." This single experiment counts for but little ; and the criticism may be made that this micrococcus was obtained from gonorrhceal pus, and is perhaps specifically distinct from the micrococcus of ordi- nary pus, although it appears to be morphologically identical with it. All this is admitted, and the experiment is introduced mainly to call attention to a method, which, carefully applied, should enable us to solve the question as to the patho- genic role of this micrococcus. The writer had mapped out for himself a series of experiments in this direction and many others relating to etio- logical questions, but circumstances have not been favorable for the prosecution of experimental work, and he finds himself, somewhat reluctantly, en- gaged in a review of the field, when it would be far more to his taste to interrogate nature by the experimental method, and thus to aid directly in the solution of these interesting problems. One of these problems, with our present light, is very puzzling. It has been demonstrated by numerous observers that this micrococcus of pus is uniformly found in pus obtained from an acute abscess, when the integument covering it is still 448 BACTERIA IN SURGICAL LESIONS. intact, even when it is deeply situated in -the tissues ; and yet the observations of Pasteur, Koch, Cheyne, and many others, are in accord as to the absence of all micro-organisms from .the blood of healthy persons. Whether, then, we suppose this micrococcus to be the cause or the result of the for- mation of these abscesses, we are met by the ques- tion, How did it get there in the first instance ? In certain cases such abscesses may be traced to an injury in which a slender, sharp-pointed in- strument— e. g., a needle or a thorn — has pene- trated deeply into the tissues ; and in this case we may suppose that micrococci have been introduced in this way. Or possibly the point of inoculation may have been far removed from the situation where the abscess is developed, and the organisms may have made their way in the blood-current or through the lymphatics to this point, where, for some mechanical reason, they have been arrested. But this is speculation, and we must leave the question unsettled, and content ourselves for the present with a summary statement of the observed facts relating to the presence of this micrococcus in collections of pus not exposed to the air. In 1875, Bergeron, in a communication to the French Academy of Sciences, reported, as the re- sult of numerous observations made for the pur- pose of ascertaining if the pus of abscesses contains bacteria, as follows : " 1. Vibrios are found in the pus of abscesses, with- out any contact with the external air, and without, BACTERIA IN SURGICAL LESIONS. 449 usually, any indication that the organism is seriously affected by their presence. 2. We cannot admit that in these cases the vibrios have penetrated into the interior of the abscess through the lymphatic system, or through the circulating system, both being intact. The pus of warm abscesses in adults often contains vibrios ; if they occur in the case of infants the fact has not been ob- served. 3. The pus of cold abscesses in the adult, as in the infant, never contains them." (Magnin.) The vibrios of Bergeron are doubtless identical with the micrococcus described by later observers, which often occurs in chains. The observations of Billroth, Cheyne, and Ogston, are in accord with those of Bergeron as to the presence of micrococci in acute abscesses, and their absence from chronic abscesses. Cheyne has shown, however, that when these organisms are proved to be present in pus from an abscess, by microscopical examination, this pus often fails to fertilize a culture-fluid, thus proving that the micrococci are no longer living. He says : " Of acute abscesses, I had up to May, 1879, inocu- lated from thirty-two cases. In twenty-five of these no growth of organisms occurred, while from six micro- cocci were obtained. In no case did I get bacteria " (I. o., p. 253). Ogston examined the pus from eighty-two ab- scesses, all of which had been "hitherto unopened." The pus was taken from them by means of a needle or a knife while still flowing from the incision, spread out in a thin film upon a slide, immediately 29 450 BACTERIA IN SURGICAL LESIONS. dried, and stained with an aniline dye. Of the abscesses examined, thirteen were — " Chronic typical cold abscesses, whose duration could be measured by months, proceeding from chronic carious disease of bone, scrofulous lymphatic glands, and such like. In none of them were any organisms found. " Four were somewhat chronic abscesses, whose dura- tion could be measured by weeks ; and which had fol- lowed diseases more or less allied to, or complicated with, forms of blood-poisoning and hectic, such as tonsil- itis, phthisis, scarlatina, erysipelas, typhoid fever, and diphtheria. All of these contained micrococci, and were evidently the same as the next form. " Lastly, sixty-five were acute abscesses, whose dura- tion could be measured by days, from all parts of the body. Every one of these contained micrococci." The cocci were found (a) in chains, usually of five or six elements, but often much longer — in one case three hundred and twenty-one cocci were counted in a single chain; (b) in groups, " like the roe of a fish" — zoogloea masses; (c) in groups of three or four, " many of which were clearly, from the equal size and relative positions of the cocci, formed by a direct division into fours, or even, though more rarely, into threes ; " (d) in some cases unusually large oval cocci were found, chiefly in pairs. " For the most part these varieties ex- isted in separate abscesses, but it frequently occurred that an abscess contained both chains and groups. Out of sixty-four abscesses where this point was specially noted, seventeen contained BACTERIA IN SURGICAL LESIONS. 451 chains only, thirty-one groups only, and sixteen both forms, or only pairs." Ogston was unable to discover any difference in the character of the abscesses which contained these different forms, and could not decide defi- nitely whether they represented different species or only varieties of the same species. To ascertain whether these micrococci possessed pathogenic properties, Ogston injected pus con- taining them into guinea-pigs and mice ; and, for comparison, pus from cold abscesses, which con- tained no micro-organisms, into other animals of the same species. The invariable result of twenty experiments, in which pus from the last-mentioned source was used, was that no illness or abscess ensued. " But a very different effect was produced when similar injections were made with pus containing micro- cocci. In every instance, with the qualifications to be presently made, well-marked disease was set up. Quan- tities, varying from one to three minims, produced, in the animals already mentioned, symptoms of blood- poisoning, lasting from two to five clays. . . . These symptoms became less marked towards the end of the first week or five days. If the animal was killed during this stage, the blood in its right heart was found to con- tain micrococci ; single, in pairs, and in short chains of six or fewer, swimming in the serum between the cells. Around the site of injection was found a patch of red infiltration, varying in size, and having in its centre more pus than corresponded with the quantity that had been injected. The pus contained myriads of micrococci 452 BACTERIA IN SURGICAL LESIONS. of the same nature as those injected, but more numer- ous. . . . The cocci were living and growing, and a drop of the matter injected into another animal pro- duced the same results in it, and it on another animal, and so on. No increased virulence was observable in the transference through a series of animals. The red infiltration around the abscess showed the micrococci invading the neighboring tissues, penetrating between their cells, and in colonies or chains, gradually decreas- Fig. 30. Group of chain micrococci in pus. x 1600 (Ogston.) ing in size, pushing their way for a considerable distance into the structures in the vicinity. . . . After five to seven days had elapsed, and in some cases even earlier, the animals exhibited a change. They became more active again, threw off their lethargy, and seemed well ; but at the spot where the injection had been made, there was found a fluctuating tumor, gradually increasing in size, and presenting all the signs of being an ordinary abscess. When they were killed during this second stage, micrococci were more rarely found in the heart- BACTERIA IN SURGICAL LESIONS. 453 blood, and the infiltration of the organisms into the tissues around the abscess no longer existed, having been replaced by a firm, thick wall of granulation tissue, in which micrococci could seldom be detected, and which seemed to act as a barrier, preventing or diminishing their migration into the blood and surrounding structures. . . . " On the presumption that carbolic acid would de- stroy the power of the micrococci, a series of injections were instituted with pus mixed with equal parts of a five per cent watery solution of that substance. These were employed on separate animals, as well as on a different part of an animal injected with unmixed pus from an acute abscess ; and in every case, the pus so disinfected, though injected in larger quantity, produced no reaction whatever, but disappeared in the rapid and complete way described under the experiments with that from cold abscesses. " I next endeavored to ascertain the temperature capable of destroying the power of micrococci. Although, in this direction, the experiments were not so numerous as is desirable, it may be stated that pus heated to 130° Fahr., or higher, hitherto always failed to excite suppuration." This is strong evidence in favor of the view that the formation of acute abscesses is due to the presence of this micrococcus. The writer has shown that its thermal death-point is 140° Fahr., the time of exposure being ten minutes. Ogston does not state the time of exposure to a tempera- ture of 130° Fahr., but it may well be that a somewhat longer exposure than ten minutes at this temperature would also be fatal to the mi- crococcus. 454 BACTEIUA IN SURGICAL LESIONS. Ogston made also a large number of culture- experiments. " As might have been anticipated, cultivations of pus of cold abscesses (five cases) yielded uniformly negative results. " Cultivations of pus of acute abscesses gave at first the most inexplicable and contradictory results. This was ascribed to the fact that the micrococcus in question is anaerobic, and cannot grow in the presence of oxygen. The plan was therefore tried of growing them in eggs. Newly-laid eggs were washed in five per cent carbolic water ; and, under spray, a minute aperture was pierced in the larger end. One minim of pus from an acute abscess, collected under the strictest antiseptic precautions, was injected by a long-pointed pure syringe into the albumen at the opposite end of the egg. A piece of protective was laid over the aperture. The egg was enveloped in a Lister's dressing, and kept for ten days in the incubator at 98° Fahr. At the end of that time it was opened, and my expectations were fulfilled. The egg was sweet and fresh ; its contents were unaltered, save the yolk was somewhat broken up, and more or less mixed with the albumen ; but the albumen, and sometimes the yolk also, were filled with enormous chains or masses (accord- ing to the sort of coccus used) of micrococci, growing quite as luxuriantly as I had ever observed them when experimenting on animals. A drop of the albumen in- jected into an animal's back now produced typical abscess, with all the symptoms already mentioned ; and the animal, on being killed, showed the micrococci in the blood and invading the tissues, exactly as had been already obtained by the employment of the pus of acute abscesses." This is an extremely interesting experiment, BACTERIA IN SURGICAL LESIONS. 455 but Ogston is evidently mistaken in ascribing the contradictory results at first obtained to the fact that the micrococcus in question is anaerobic ; for while this is true, and it can doubtless grow in the absence of oxygen, the writer has found no diffi- culty in cultivating it through successive gener- ations, in the culture-flasks described on page 177, in bouillon made from the flesh of a rabbit or of a chicken, and in the presence of atmospheric air, with which these flasks are two-thirds filled when prepared in the manner indicated. Thus, in my experiments upon the germicide power of various therapeutic agents, a pure-culture of this micro- coccus was maintained through many successive generations, culture No. 1 having been inoculated with a drop of pus from a whitlow, obtained at the instant of its escape from a deep incision. The true explanation of the contradictory results ob- tained by Ogston is doubtless that given by Cheyne, viz. : that when no development occurred in cul- ture-solutions inoculated with the pus of acute abscesses, it was because the micrococci were already dead. Wernich has shown that during the multiplication of various bacterial organisms in a limited amount of nutritive pabulum, chemical products are evolved fatal to the vitality of these organisms. In conclusion, the writer would suggest that those who desire to make themselves familiar with the organisms to which a pathogenic role has been 456 BACTERIA IN SURGICAL LESIONS. ascribed, and with the technique relating to their recognition, cultivation, etc., will do well to com- mence with this micrococcus of pus, a pure culture of which may be easily obtained in the manner heretofore indicated. BIBLIOGRAPHY. ALBRECHT, R. — Beitrag zur Kenntniss und Entwicklung der Spirochsete Obermeieri. Deutsches Arch. f. Klin. Med., Leipz., 1881, XXIX. 77-112. ALMQUIST, E. — Tyfoidfeberns bakterie. Nord. med. Ark., Stock- holm, 1882, XIV. No. 10, 1-31, 1 pi. . Method of cultivating and coloring bacteria. Hygeia, Stockholm, 1883, XLV. 22(3-262. ANDRAL and GAVARRET. — Recherches sur le mode et les circon- stances de developpement d'un vegetal microscopique dans les liquides albumineux, normaux et pathologiques. Gaz. ined. de Par., XI. 87-91. 1843. 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Nature de 1'immunite des moutons algeriens centre le sang de rate; est-ce une aptitude de race? Ibid., XCI. 33-36. . De I'attemiation des effets des inoculations virulentes par 1'emploi de tres petites quantites de virus. Compt. rend. Acad. d. sc., Par., 1881, XCII. 844-848. Etude des ferments et des virus. Gaz. d. hop., Par., 1881, LIV. :}.-):]; :\(>\. . Etude experimentale des conditions qui permettent de rendre usuel 1'emploi de la methode de M. Toussaint pour attenuer le virus charbonneux et vacciner 1<^ ;inimales sujettes au sang de rate. Compt. rend. Acad. d. sc., Par., 1882, XCIV. 1694-1098. De rattenuation directe et rapide des cultures virulentes par Tact ion de la chaleur. Compt. rend. Acad. d. sc., 1883, XCVI. »:,: J-.-)57. — . De la faculte' prolifique des agents virulents attcnues par la chaleur, etc. Ibid. , 612-017. . Du role de 1'oxygene de 1'air dans rattenuation, etc. Ibid., 07S-OSJ, CIIAVAXXES (de Lausanne). — Les principales maladies des vers a soie. 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WOOD and Foft&tAD. — Report upon the effect of inoculating the lower animals with diphtheritic exudation. Rep. Nat. Bd. Health, 1880. Wash., 1882, II. 374-386. WOBTMANX, J. — Diastatic Ferment of Bacteria. Zeitschr. f. phys.-Chim., VI. 287. WYSS, OSCAH. — Ueber typhose Erkrankungen, durch Fleischgenuss Correspondenzblatt der Schweiz, Aertze, XI. Jahrgang, 1881. ZIEGLER. — The Schizomycetes, or Bacteria. Chap. XXX. in Text- book of pathological anatomy and pathogenesis. London and New York, 1883 ZWEIFEL, P. — Untersuchungen iiber die wissenschaftliche Gruml- lage der Antisepsis und die Entstehung des se})tischen Gifts. Ztschr. f. physiol. Chem., VI. 380-121. Strassb., 1882. INDEX. ABSCESSES, bacteria in, 449. Acetic acid, antiseptic action of, 215. Acetic ferment, 83. Aerobics, 116. Aeroscopes, 200. Alcohol, germicide power of, 215. Algae, bacteria classed with, 56. Aluminium acetate, 216. Aluminium chloride, 216. Ammonia, does not dissolve bac- teria, 54. germicide power of, 216. source of, 149. Anaerobies, 116. Anthrax, 265. bacillus of, 270. spores of, 270. Aqueous humor as a culture-fluid, 165. Aromatic products of decomposition, 216. Arsenious acid, 216. Atmospheric bacteria, collection of, 197. Attenuation of vims : method of Pasteur, 202. of Toussaint, 204. of Chauveau, 205. by intravenous injection, 206. by chemical reagents, 206. BACILLUS, 87. B. anthracis, 88. development of, from B. subti- lis (/), 253. Bacillus malaria, 319. action of quinine upon, 327. in blood of man, 320. Bacillus of leprosy, 332. of malignant oedema, 336. of tuberculosis, 391. of typhoid fever, 408. B. amylobacter, 88. B. des infusions, 90. B. du levain, 89. B. du vin tourne, 90. B. glaireuse, 90. T>. intestinal, 89. B. ruber, 89. B. subtilis, 87. B. utilis, 89. Bacteria in surgical lesions, 442. Bacterium, 80. genus established by Davaine, 18. B. ffiruginosum, 85. B. catenula, 82. B. cyaneum, 73. B. lineola, 81. B. littoreum, 81. B. luteum, 73. B. prodigiosurn, 73. ' B. ptmctum, 82. B. termo, 81. B. sulphuratum, 86. B. violaceum, 74. B. xanthinum, sr>. Bacterio-purptirine, 38. Bastian, views and experiments of, 103. 490 INDEX. Baumgarten, method of staining tubercle bacilli, 191. Beggiatoa, 91. B. alba, 91. B. arachnoidea, 91. B. leptomitifornris, 91. B. minima, 91. B. mirabilis, 91. B. nivea, 91. Benzoic acid, antiseptic value of, 217. Pert, experiments of, 269. Billroth, views of, 22. Blood, normal, free from bacteria, 108, 261. method of obtaining, 161. Blood-serum as a culture-medium, 162. method of sterilizing, 163. Bockhart, experiments of, 311. Pokai. experiments of, 310. Boric acid, antiseptic value of, 122, 217. Bromine, germicide power of, 218. CAMI-HOU, antiseptic value of, 218. Carbolic arid, action of, upon bac- teria, 122, 219. Carl ion, how obtained by 1- 113. Carbonic; field, action of, upon bac- teria, 122. Cell-membrane of bacteria. • ro-spinal menin Ci'ii, investigations of, :'>-_V>. Characters, generic and specific, 60. Charbon, 5 Chiirhon symptoniati. M. crepusculum, 75. M. cyaneus, 7-3. M. diphtheriticus, 76, 292. M. fulvus, 74. 492 INDEX. Micrococcus luteus, 73. M. of epidemic diarrhcea, 77. M. of exarithematous typhu^ 78. M. of glanders, 78. M. of intestinal typhus, 78. M. of pyeemia of rabbits, 344. M. of rugeola, 77. M. of scarlatina, 77. M. of septicsBrnia of rabbit, 359. M. of stringy wine, 75. M. of syphilis, 78. M. of the variola of animals, 77. M. prodigiosns, 73. ]\1. scpticus, 70. M. im-re, 75. M. vaecime, 76, 412. M. of variola, 441. M. violacens, 74. Micrococci in measles, 341. in pus, 304. in gonorrhoea! pus, 301. in erysipelas, 2 in wounds treated aseptically, 444. ill acute abscesses, 447. Microsphrcra vac,cin;e, 76. Microsporon scpticus, 76. Miero/yma bombycis, 76. Milk as a culture fluid, 163. Milk sickness, :^39. Miltzbrand, Mi. M. Warmingii, 79. Movement, brownien, 33, (•••! use of, 34. of two kinds, 32. Miiller, 0. F., classilifation of, 14. Multiplication, rapidity of, 125. lerma, form of, 45. ;i, 83, 140. M. vim, 141. Myconostoc, 96. M. gregarium, (J7. NAGELI, classification of, 57. Nitrification, role of bacteria in, 149. Nitrous acid, 226. Nitrogen, how obtained by the bac- teria, 112. Nutrition of the bacteria, 111. OGSTON, experiments of, 449, 454. Oil of mustard, 226. Oil of turpentine, ^i2»>. Ophidomonas sanguiuea, 80. Origin of the bacteria, 101. Oscillaria malaria? of Laveran, 329. Osinic acid, 226. Oxnlic acid, 22(5. Oxygen, rule of, 115. germicide power of, 227. Ozone, action of, upon the bacteria, 121. germicide power of, 227. P.U.MKMA prodigiosa, 73. Panum, experiments of, 262. Pasteur, experiments relating to hy- drophobia, 314. Pathogenes, 7".. Pathogenic bacteria, development of, '2.', ± Penicillinm, in blood of yellow fever, 426. Pernicious fever, 325. Petalohacteria, 46. •occos, 46. • •aphing bacteria, 194. Picric acid. Pigmentary bacteria, 7_. of the bacteria in vegetable Pleura-pneumonia, 341. Polymorphism, 133. ii of the bacteria, 48. ::iicide value of, 227. Potassium arsenite, 228. chlo iodide, 228. INDEX. 493 Potassium nitrate, 228. permanganate, 228. Protective inoculations in anthrax, 277. in septicaemia, 372. modus operandi, Pasteur's ex- planation of, 242. Author's explanation, 246. Protoplasm, currents in, 37. granules in, 37. of the bacteria, 36. Pseudobacteria, 50. Ptomaines, 257. Pulverulent precipitate, consisting of bacteria, 46. Pure cultures, methods of obtaining : Lister's method, 156. Koch's method, 157. Pus, bacteria in, 111, 307, 444. Putrefaction, role of bacteria in, 148. Pyaemia in rabbits, 343. Pyrogallic acid, 229. QUININE, germicide value of, 229, 326. RECOGNITION of bacteria, 184. Relapsing fever, 346. inoculation experiments relat- ing to, 347. Reproduction of the bacteria, 123. by fission, 123. by spores, 126. Respiration of the bacteria, 111. Rhabdomonas rosea, 80. Rosenberger, experiments of, 259. SACCHAHOMYCETES, 57. Sachs, classification of, 57. Salicylic acid, 2-29. Saliva, micrococcus of, 359. Sarcina, 96. Scarlet fever, 349. in animals, 350. Schizomycetes, Xiigeli, 57. Schizophytes, Colin, 66. Sepsin, 258. Septic toxaemia, 259» Septicaemia in mice, 351. Septicaemia in rabbits, 355. etiology of, 356. pathology of, 360. Soda, germicide value of, 230. Sodium biborate, 231. Sodium chloride, 231. Sodium hyposulphate, 232. Sodium salicylate, 232. Sodium sulphite, 232. Species, physiological, of Pasteur, 63. value of, 61. Spherobacteria, 71. Spirillum, 94. S. attenuatum, 96. S. Rosenbergii, 96. S. rufum, 94. S. tenue, 94. S. undula, 94. S. violaceum, 96. S. volutans, 94. S pi ro bacteria, 91. Spirochsete, 93. S. gigantea, 93. S. Obermeieri, 93. S. plicatilis, 93. Spirochaete Obermeieri, 347. Spiromonas Cohnii, 80. Sporangia, 130. Spores, development of, 128. germination of, 131. Spores of B. anthracis, 270. Spreading abscess in rabbits, 376. Staining bacteria, 186. Starch, in Bacillus amylobacter, 39. Sterilization of culture-fluids, 168, 172. Structure of the bacteria, 35. Sulphur, contained in bacteria, 33. Sulphuretted hydrogen, 234. Sulphuric acid, 233. Sulphurous acid, 233. Swine plague, 378. Symptomatic anthrax, 280. Syphilis, 380.' Syphilitic organisms, 381. 494 INDEX. TAXNIC acid, 234. Temperature, action of, upon bac- teria, 118. Thermal death-point of bacteria, 119. of 13. anthracis, 27o. of M. of fowl cholera, 289. of septic inicrococcus, 364. Thermostat for gas, 181. electro-magnetic, 183. Thymol, '2:34. Tomma.si-Crudeli, experiments re- lating to intermittent fever, 319, 325. Torula form of bacteria, 43. Tubercle bacillus, 386. ni(5l hods of staining, 190. morphology of, 393. Tubercles without bacilli, 390. Tuberculosis, 384. Typhoid bacilli of Klebs, 407. of Eberth, 409. Typhoid fever, 400. lower animals not subject to, 403. K endocarditis, 411. Urine as a culture-fluid, 104. method of obtaining free from bacteria, 165. VARIOLA, 411. Variola of pigeons, 413. Venom of serpents, 261. Vibrio, 92. V. bacillus, 89. V. lactic, 83. V. lineola, 81. V. prolifer, 94. V. rugula, 92. V. serpcns, 93. V. subtilis, 87. V. syncyanus, 85. V. synxanthus, 85. V. tartaric right, 83. V. treinulans, 81. Vibrioniens, definition of, Ehren- berg, 16. it, examination of, 201. Whooping cough, 415. Wood, 11. C., experiments of, 295. Yr.i.r.mv fever, 417. blood of, 4-J-J, 108. Yellow-fever commission, extracts from report of, 421. Yellow-fever germ of Carmona, 432. of Freire, 4^9. /INC chloride, 234. Zinc sulphate, 235. i form of grouping of the bacteria, 44. Zoogbea, genus established by Colin, 21. 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