o: x. : tr =o o r-q ru o O CD m THE STRUCTURE AND FUNCTIONS OF BACTERIA HENRY FROWDE, MA. PUBLISHER TO THE UNIVERSITY OF OXFORD LONDON, EDINBURGH, AND NEW YORK THE STRUCTURE AND FUNCTIONS OF BACTERIA ALFRED FISCHER PROFESSOR OF BOTANY AT THE UNIVERSITY OF LEIPZIG TRANSLATED INTO ENGLISH 73 Y A. COPPEN JONES (Djforfc AT THE CLARENDON PRESS 1900 PRINTED AT THE CLARENDON PRESS BY HORACE HART, M.A. PRINTER TO THE UNIVERSITY / o AUTHOR'S PREFACE SEEING that there is at the present time no lack of treatises upon bacteria, the publication of yet another needs some apology. Such an apology is expressed in the title, Lectures on Bacteria *. The lectures are intended to be an introduction to general bac- teriology. They purpose to give a survey that shall collect and condense the innumerable special researches into a connected whole, and indicate in broad outlines the present position and extent of bacteriological science. Besides those medical aspects of bacteriology which in other treatises occupy, rightly enough, the chief place, attention is drawn to the importance of bacteria in agriculture, and to the parts they play in the great fundamental processes of life — the circulation of nitrogen and carbon dioxide. Furthermore, it seemed desirable to point out and emphasize the advancement that general physiology has received from bacteriological investigations. Finally, an attempt has been made to remove the bacteria from the isolated position to which their morphological and physiological pecu- liarities had relegated them, and, by comparative studies, to indicate their relations to other organisms. A treatment of the subject on these lines that should be at the same time not too bulky, seemed to me to be wanting. I there- fore undertook the publication of the course of lectures I have delivered for some years to students of biology, pharmacy, and agriculture, with here and there among them — as it were like a white raven ! — a medical student. ALFRED FISCHER. LEIPZIG, 22 July, 1897. The title of the German work is Vorlesungen uber Bakterien. TRANSLATOR'S PREFACE IN offering to English readers a translation of Professor Alfred Fischer's Vorlesungen iibcr Bakterien, an Introduction is rendered almost unnecessary, in the first place by the favourable reception of the original German edition, and secondly, by the fact that no work on Bacteriology of similar scope and mode of treatment has appeared in England since De Bary's classical Lectures on Bacteria. But those Lectures were published in 1887, and the twelve years that have passed since then have been years teeming with activity in every field of bacteriological research. The present work epitomizes, in a comparatively small space, the results of these investigations, and attempts to elucidate them in their relations to the many-sided problems of Pathology, and of Technical and Agricultural Chemistry, and the great chemical processes of nature. It should be stated that the few variations from the original that occur have been made with the Author's sanction, and that, furthermore, the book has enjoyed the advantage of a proof- revision by Professor Marshall Ward of Cambridge. The Index is the work of Dr. Alfred J. Ewart. A. C. J. DAVOS PLATZ, March 13, 1900. CONTENTS CHAPTER I TAGE INTRODUCTION : MORPHOLOGY Form, Size, and Structure of the Bacterial Cell, Cell-membrane and Cell-contents . i Morphology of the Bacterial Cell . . ...... 2 Finer Structure of the Bacterial Cell . 4 CHAPTER II MORPHOLOGY (continued} Pigments, Intracellular Products ; Movement and Organs of Locomotion ; Cell-division ; Spore- formation and Germination ... . . ..12 Movements and Organs of Locomotion . 14 Reproduction of Bacteria by Fission . .16 Spores and Sporulation .... 19 CHAPTER III TAXONOMY The Question of ' Species ' among Bacteria ; Variability ; Involution and Attenuation ; the Classification of Bacteria .... . 24 CHAPTER IV TAXONOMY (continued') The Systematic Position of Bacteria ; Other low Organisms with Pathogenic Properties . . 35 CHAPTER V DISTRIBUTION AND ORIGIN Distribution of Bacteria ; Their modes of life ; Spontaneous Generation . 44 CHAPTER VI PHYSIOLOGY OF NUTRITION AND GENERAL PRINCIPLES OF CULTURE Chemical Composition of Bacteria . 52 Food Stuffs of Bacteria . . 53 CHAPTER VII RESPIRATION OF BACTERIA Aerobiosis and Anaerobiosis ; Light-producing Bacteria ; Marine Bacteria ; Sulphur and Iron Bacteria . ....... .60 CHAPTER VIII INFLUENCE OF PHYSICAL AGENTS Light, Electricity; Pressure, Temperature, Dryness and Moisture; Disinfection by means of Physical Agents ..... . . • 71 CHAPTER IX THE ACTION OF CHEMICALS Chemotaxis and Chemical Disinfection . . .- . 78 viii CONTENTS CHAPTER X PAGE BACTERIA AND THE CIRCULATION OF NITROGEN IN NATURE 1. Introduction: The Assimilation of Free Nitrogen by the Bacteria of the Root Nodules of Leguminous Plants, and by Bacteria in the Soil ........ 88 CHAPTER XI BACTERIA AND THE CIRCULATION OF NITROGEN (continued) 2. The Liberation of Organic Nitrogen by Putrefaction, and its Mineralization by the Nitri- fying Bacteria .............. 98 CHAPTER XII THE CIRCULATION OF CARBON DIOXIDE IN NATURE 1. Introduction; Organized Ferments and Enzymes ; Races of Ferment Organisms; Fermen- tation of Alcohol and of Acids; Optical Activity and Fermentation .... 107 CHAPTER XIII THE CIRCULATION OF CARBON DIOXIDE IN NATURE (continued} 2. Bacterial Fermentation of Carbohydrates . . ...... 116 Milk and other Dairy Products . . . . . . . . . . . 117 Butyric Fermentation ..... .......121 Cellulose Fermentation ....... 122 Mucilaginous Fermentation . . . . . . . . . . . .123 Fermentations in technical processes . . . . . . . . . .123 CHAPTER XIV THE CIRCULATION OF CARBON DIOXIDE IN NATURE (continued} 3. The Yeast Fungi and Alcoholic Fermentation. Theory of Fermentation and Anaerobiosis. Concluding Remarks on the Circulation of Nitrogen and Carbon in Nature . . .126 Theory of Fermentation and Putrefaction .......... 133 CHAPTER XV BACTERIA IN RELATION TO DISEASE 1 . Diseases of Plants ; Harmless ' Messmates ' in the Human Body ; Pathogenic Bacteria ; Points of Attack and Sources of Infection 137 CHAPTER XVI BACTERIA IN RELATION TO DISEASE (continued} 2. Description of sorr.e Pathogenic Species 147 CHAPTER XVII BACTERIA IN RELATION TO DISEASE (continued) 3. Mode of Action of Bacteria, and the Reaction of the Organism. Serum Therapeutics and Immunity ............... 158 NOTES 169 INDEX 185 The Arabic figures inseited in round brackets in the text refer to the Bibliographical Notes, pp. 169-184. CHAPTER I INTRODUCTION: MORPHOLOGY Jo "J Form, Size, and Structure of the Bacterial Cell, Cell-membrane, and Cell-contents. THE first historically recorded discovery of bacteria was made more than two hundred years ago by Anton van Leeuwenhoek, the Dutch naturalist and pioneer in the world of the infinitely little. Exploring those then untravelled regions with home-made lenses of wonderful efficacy, he found in the saliva of the human mouth minute organisms to which he gave the name of animalcula on account of their power of movement. In the description (1) and in the figures he gives (reproduced in facsimile in Fig. i), curved and straight forms and long and short rods are plainly recognizable. They constitute the earliest reliable record of bacteria, the study of which in later times has revolutionized medicine and expanded into a new science. From the year 1683 Leeuwenhoek 's observations stood alone until, a century later, the Danish savant Miiller made further investigations on bac- teria. He classified them with the infusoria and gave them names that are now familiar to us all, Vibrio^ Spirillum, and Bacillus. In 1838 Ehrenberg described in his great work on infusoria a large number of bacterial forms, ranking them with his group Vibrionia, and from this time forward the bacteria have never again drifted entirely out of sight. It was not, however, until about the seventh decade of this century that FISCHER 1'. FlG. I. Oldest known figures ot genuine bacteria (bacteria of the mouth) from Leeu- wenhoek. A and f represent Bacillus hue- calls -niaxintus. B is perhaps Vibrio buccalis ; its movements were followed by Leeuwenhoek from Clo D. E\sa. species of coccus, and G, no doubt, a Spirillum spiiti- gctnini (compare with FlG. 26). 2 INTRODUCTION: MORPHOLOGY the subject was taken up by pathologists, who from that time to this have contributed more than any others to the building up of the science of bacteriology. It was after the publication of Robert Koch's (2) first studies on anthrax that the activity of those numerous investigators began, whose incessant industry has so furthered our knowledge of these most minute organisms and has accumulated a bulk of material such as the most voluminous treatises (3) are inadequate to grapple with. Before this period of expansion and rapid growth, a growth that has been in some directions too luxuriant for strength or permanence, the bacteria had been studied chiefly by botanists (Cohn, Nageli). Their work had been both physiological and structural, and it is on the foundations which they laid that the science in its modern form has been built up. For the fundamental labours of these earlier years, that saw, too, Pasteur's brilliant researches into the physiology of fermentation, the student may be referred to Loffler's lectures on the history of bacteriology (4). Morphology of the Bacterial Cell. The vegetative phase of all the smaller bacteria consists of a single cell. In the simplest types this has the shape of a sphere (coccus}. If one diameter is greater than the other, i. e. if the sphere becomes a cylinder, it is spoken of as a rod or bacillus. In one group of cylindrical bacteria the cells are more or less spirally twisted ( Vibrio, Spirillum, Spirochaete}. In Vibrio (Fig. 2, c) the twisting comprises only a quarter of a revolution or less, in Spirillum (Fig. 2, a) one or more widely separated turns, while Spirochaete (Fig. 2, e] exhibits numerous closely-wound spirals like those of a corkscrew. The simplest method of permanently fixing the shapes of the bacteria is to let a small drop of water containing them dry upon a cover-glass. By this means they are all flattened to the plane of the glass, the vibrio, for instance, appearing, in spite of its three-dimensional curvature, only as a slightly curved 'comma'-shaped rod (Fig. 2, d}. It was for this reason that Koch called the vibrio of Asiatic cholera the comma-bacillus, but as a matter of fact it has, apart from the curvature, no resemblance to a comma. A spirillum dries in the form of a semicircle (Fig. 2, b] and a spirochaete appears as a sinuous line (Figs. 2,e; 26, f). The spirochaetes often attain great length, but it is uncertain whether they then consist of a single cell or of a connected chain of several cells. The other types of bacteria — cocci, bacilli, vibrios, and spirilla — are always unicellular. They may be grouped together as Haplobacteria as contrasted with the many-celled true filamentous forms, the Trichobacteria. In the members of this group (in the sulphur-bac- terium Beggiatoa (Fig. 17, a\ for instance) the vegetative phase is an un- branched chain or filament of closely united cells, each one having the shape of a bacillus and becoming free from the others and motile at the period of MDltrHOI.OGY OF THE BACTERIAL CELL reproduction. The unbrancJicti filamentous bacteria without definite sheaths are generally classed too-other under the collective name of LcptotJirix. The most complex form is CladotJirix, an aquatic genus with numerous dicho- tomous branchings. Here the cell-chains arc enclosed in a separate sheath and the lateral shoots arise by single cells pushing sideways through this envelope and growing out in a new direction. The resulting branch is thus only superficially connected with the parent stem (Figs. 2,/; 12). This is spoken of as ' false branching ' as contrasted with true branching, such as occurs in the mycelium of a fungus (Fig. 2,g\ In this case one of the cells of the main stem sends out a lateral cvagination which, continuing to grow in the new direction, forms a branch. This new member is a lateral oitt- groivth of iJie parent cell itself, and as intimately connected with the cells of the stem as they are with one another. Among the tricho- bacteria this method of growth is \j unknown. We have now enumerated the various morphological types occur- ring among the bacteria, and there remain to be considered the aggre- gates and colonies which arise among the haplobacteria by the multiplication of the single cell or the proliferation of large numbers in close proximity. The Anthrax bacteria, for instance, often form unbranched threads or chains which are externally undistinguishable from those of the true trichobacteria (Fig. 28). Unlike these, however, the anthrax filament may at any time break up into its constituent cells irrespectively of the advent of the repro- ductive process. Short threads of two or three cells, as well as single cells, may also occur. These phenomena, both in bacilli and cocci, will be con- sidered in more detail when the question of species is under discussion. Very frequently the bacterial cells are found collected in masses of more or less regular contour held together by a gelatinous substance. To these masses the name of Zoogloea has been given. They occur both in liquids and on the surface of solid nutritive substances, such as potatoes or gelatine. Sometimes in liquids they form a skin or pellicle composed of very dense FlG. 2. fl, Spirillum uiidula. living, with spiral twisting ; b, the same dried on the cover glass in semicircular shapes. c, Vibrio cholerac, slightly spiral ; d, dried in comma form. et Spirochaele Obermaieri from the blood in recurrent fever. _/, Cladothrix dichotoma^ a branched example, with sheath and so-called ' false branching ' ; abovey~a short branch of two cells is just pushing through the sheath, g, Penicilliuiii glaucum, a fragment of mycelium wilh true branching (from Brefeld). Amplification : a and b 1500, c and d 2250, e about 8oo,y"6oo, g 120. INTRODUCTION: MORPHOLOGY aggregates of individuals. In many cases the zooglocae and pellicles are merely social aggregates of individuals*, just as a forest or a meadow is, and, like these, cannot be considered as morphological units. In others, such as the cloud-like mass shown in Fig. 3, or the delicate ramifying growth of B. proteus (Fig. 22), we have a true 'colony' before us, whose shape is not accidental, but a result of definite modes of growth and multiplication which recurs regularly in every culture, the characters thus produced being often of taxonomic value. In all such zoogloeae and pellicles each single bacterium is independent of the others. The plant remains a single cell, and there is never any division of labour such as occurs in the more complex colonies of the lower plants (volvocineae) or animals (coelenterata). The bacteria are the smallest of all known organisms, the most bulky coccus having a diameter of only about 2^c (-j-^j mm.). ju ,f% •:' '«•.'•*'-...: >v ;"-' • W • ;. ^ FIG. 3. Fragment of the botryoidal zoogloea of an aquatic bacterium (Zoo- gloea ramiirera) of older authors. The rods are thickly aggregated at the peri- phery, less so in tne middle ; they are held together by mucus. Magn. 56. Compare the zoogloeae in Figs. 17, e, and ~ Among the Staphylococci (the most widely distributed pus-bacteria) the diameter is often not morethano-8/z.and the volume accordingly only ! 700-000^00 CUD- mm- Inasmuch as the cell-substance contains a large proportion of water, the weight, too, is inconceivably small ; thirty billion would weigh only a gram. In a drop of water i c. mm. in size, seventeen Jiundrcd million pus-cocci would have room and to spare. Even the comparatively large anthrax bacillus is only 3-io/x long and i-i-2/u, broad, so that its volume would have to be increased eight million times before it attained the bulk of a middling- sized cigarette. Finer Structure of the Bacterial Cell (5). It would seem at first sight a hopeless task to attempt to gain an insight into the architecture of so minute a thing as a bacterium. Nevertheless the problem has been attacked, and, thanks to the efficacy of modern micro- scopes, some new facts have been established. We might well suppose that these organisms, standing as they do upon the very threshold of life, would be of simpler structure than those elementary units, the cells, of which all the higher plants and animals are built up. The first question then to be answered is whether all those parts which constitute a simple cell, such as a plant-cell, The word ' growth-form ' is often applied to these aggregates. This literal translation of the German Wuchsform is an unfortunate one, inasmuch as the words growth and Wuchs do not connote precisely the same ideas, and, furthermore, the expression is used by different authors in slightly different senses (cf. Goebel, Outlines of Special Morphology of Plants, p. 480). — -^ can be distinguished in the bacteria. The parts arc : the cell -wall (if, Fig- 4, rt), the protoplasm (/>, Fig. 4, a], the nucleus (k, Fig. 4, «), and the cell-sap (.r, Fig. 4, a). The cell-sap is contained cither in spaces (vacuolcs) scattered through the protoplasm, or in a large central cavity that fills out the greater part of the cell, the protoplasm being reduced to a thin layer coating the cell-wall (primordial utricle}. Now the relation of this protoplasmic lining of the cell to the cell-wall is of great importance in the study of the structure of bacterial cells, and it will be necessary to discuss in some detail the conditions present in the case of plant-cells in general. It will then be seen how far the observable facts are competent to elucidate the finer structure of bacteria. The cell-sap of vegetable cells consists of water in which are dissolved various substances, organic compounds and mineral salts. The protoplasm, which forms a sac enclosing the cell-sap, is during life endowed with properties similar to those of animal membranes, being readily permeable to pure water, but almost or quite imper- meable to the substances held in solution by the cell-sap. As a result, these bodies exert a strong pressure (osmotic pressure] from within outwards upon the proto- plasmic sac, stretching it and pressing it tightly against the comparatively un- . yielding Cell-Wall. An explanation Of this is afforded by the modern Theory * Of Solutions which shoWS that the mole- CUleS Of a substance dissolved in water 2-5 /0 INat-l ; the contents form two separate act as though they were in a gaseous spherical masses. Magn. 3«). state and strive to fly outwards a\vay from each other. Consequently, if the membrane is in the form of a closed sac surrounded by pure water and containing a solution, the substance dissolved behaves as a gas, and its molecules, like those of hot air in a bladder, expand and dilate the sac until the pressure inside is equal to that without. In the case of the protoplasmic sac of plant-cells this condition of equilibrium is unattainable, because unlimited dilatation is prevented by the cell-wall, and the result is that the whole of the cell is during life in a permanent condition of strain (turgor}. It will be observed that osmotic pressure can only originate when there is water on both sides of the membrane, and this condition is present not only in water- plants, but also in terrestrial vegetation, inasmuch as the cell- walls are always saturated with water. If we now reverse the conditions and, instead of placing the cell in pure water, lay it in a solution of greater osmotic pressure than that of the cell-sap (5 per cert, saltpetre or 2-5 per cent, common salt) FIG. 4. riasmoiysis Of a ceil from a fine hair of Ecballiitni elateriiim. a, distribution of the cHl-rontents in natural state Amounted in water); HI, cell-wall; />, protoplasm (primordial utricle); j, cell-sap in central vacnole;*, nucleus. £,in2-5% common salt solution, medium degree of plasmo- bsis; th,e .protoplasm has retreated from the cell- wall and is being constricted into two parts, c, after lying about half an hour in the 6 INTRODUCTION: MORPHOLOGY it is easy to foresee what will happen. The pressure on the outside of the protoplasmic sac being now greater than that within, the protoplasm is pressed inward away from the cell-wall. The sac shrinks until the concentra- tion of the cell-sap is such that the osmotic pressure it exerts is just equal to that of the fluid outside. Since the shrinking of the protoplasm commences directly the pressure of the surrounding medium exceeds that of the cell- sap, we have in this phenomenon, which is known as Plasmolysis, a means of measuring the pressure within the cell. The gist of the whole matter lies in the fact that the protoplasm is easily permeable to water both within and without, but presents a more or less impassable barrier to the molecules of the osmotic substances. As a result, the cells may lie for a long time in the plasmolysing solution without returning to their original state. If they be removed from the solution and placed in pure water again, the pressure of the molecules inside the protoplasm immediately reasserts itself, and the sac dilates until the original condition of turgescence is restored. All these phenomena are only observable in the living cell. At death the protoplasm loses its impermeability, and presents no hindrance to the molecules of substances in solution. In spherical cells the protoplasm is contracted by plasmolysis to a ball, but in long cylindrical cells, such as hairs for instance (Fig. 4), or algal cells, the elongated sac generally becomes constricted into two or three portions. These are at first connected by strands of protoplasm (Fig. 4, b), but finally separate and form distinct masses. The commonest case is where two round or oval lumps of protoplasm are left, one at each end of the cell (Fig. 4, c}. When, by transference to pure water, the process is arrested and reversed, these masses melt together again as soon as they touch and continuity is restored. This de-plasmolysation must not be too rapidly performed, or the fragments of the protoplasmic sac may burst, in which case the protoplasm invariably dies. The application of plasmolysis to the study of bacterial cell-structure may now be discussed, but it will be advisable first to consider the results which other methods of investigation have yielded, notably those of fixing and staining. If we examine bacteria under a very high power objective (2,000 diams. and over) very little can be seen. The cell has a fairly sharp outline, but it is not possible to distinguish a separate membrane enclosing the cell-contents. The latter appears as a pale homogeneous mass with occasional granules of stronger refringency, and in the case of some of the larger bacteria (Spirillum, Cladothrix] sap-vacuoles differentiate themselves from the protoplasm by their watery appearance. These meagre details are all that can be seen in the fresh unprepared state, — of the organs of locomotion nothing can be seen, — but by the customary methods of fixing and staining a number of other particulars can be made visible. If a minute speck of a pure culture be mixed with a fixing fluid (e.g. osmic or chromic acid), spread upon a cover-glass, and allowed to dry, the bacteria adhere 5/Vvvr/rAvr or THE BACTERIAL CELL strongly to the glass and may by washing be freed from the fixative and stained with hacmatoxylin or an anilin dye. Under this treatment all bacteria, save the most minute forms, show the same structure (Fig. 5)- The membrane appears as a sharp outline enclosing a protoplasmic mass which is smooth and homogeneous where it lines the membrane, but full of irregular spaces (vacuoles) towards the centre. The protoplasm is uniformly stained and shows no trace of finer details. Only the strongly refracting granules already referred to take on a deeper tinge. Being similar in this respect to the 'chromatic substance' of cell-nuclei, they are sometimes spoken of as ' chromatin ' granules : a doubtful analogy at best. When the bacterial cell contains only one such granule (Fig. 5, a, c, d, e) it certainly makes the impression of a nucleus both as regards its size and its position in the cell. But there are, just as often as not, several granules in a single cell (Fig. 5, b, ir il- ium undula plasmolysed by the evaporation of stagnant water ; the structure of the clumps of protoplasm is well seen. Proto- plasm black in all figures. Magn. a 300, b-e 1500. 1-'L\ER STRUCTURE OF THE BACTERIAL CELL 9 tcrial cell is nevertheless considerable, namely from two to three atmospheres. In stronger solutions (~} per cent, saltpetre) the plasmolysis is rapid but ceases in a few minutes, and the protoplasm expands and fills out the cell again. This retrogression of the process is caused by the penetration of the molecules of the salt. Even in weak solutions (2-5 per cent, saltpetre) this takes place in a few hours, showing that the protoplasm of bacteria is far more easily permeable than that of the higher plants, a peculiarity which is shared by other low organisms such as flagcllata, cyanophyccac, and floridcac. A most important result of this greater permeability is the case with which bacteria are able to adapt themselves to changes of concentration in the medium they live in, and it must greatly facilitate the absorption of food- stuffs as well as the excretion of the products of metabolism such as toxins and enzyms. Finally, it must be mentioned that the motile forms retain their power of movement during plasmolysis, from which fact certain conclusions can be drawn as to the nature of the organs of locomotion. In the usual methods of preparation the liquids in which bacteria are suspended contain a considerable amount of soluble salts (the ordinary nutri- tive media hold 0-7 per cent. NaCl), and when a drop evaporates upon the cover-glass the liquid becomes more and more concentrated, so that before the bacteria become dry they are plasmolysed. It is such plasmolysed cells which we see in most cover-glass preparations, many species such as B. typhi, Vibrio cliolcrac, and Spirillum nndnla frequently showing strongly stained granules (Polar-granules) at each end of the cell, the central space being empty (Fig. 6). The meaning of these appearances is evident. The general conclusion to which all these observations lead us is that the bacterial cell-contents are a mass of protoplasm representing an osmotic system precisely like that of the cells of the higher plants, but, unlike them, having no nucleus. The membrane or cell-wall of the bacteria is in most cases thin, delicate, colourless, and without a perceptible finer structure. Unlike the cell-wall of plants, it consists of a protein substance (no doubt a modification of the protoplasm) and not of cellulose*. It would seem therefore that the degree of division of labour which characterizes the higher plants, where there is an outer very permeable membrane of cellulose, and an inner more or less impermeable protoplasmic layer, has not been reached by the bacteria where communication between the organism and the outer world is regulated by two layers of medium permeability. As is the case in many algae and cyanophyceae, the cell- wall of some bacteria secretes a jelly-like substance which appears optically as a delicate * Analysis of the cell-wall of the /). tuberculosis shows that it contains a large amount of true cellulose, so large indeed that it has been delected in tuberculous animal tissues, and was thought to be a product of metabolism (Freund;. 10 INTRODUCTION : MORPHOLOGY clear space, sometimes narrower, sometimes broader than the cell it sur- rounds (Fig. 7, c and d\ It may be rendered more visible by special methods of staining. It owes its origin to the swelling up of the outer layers of the cell-wall by absorption of water, the inner layers being con- stantly built up anew from within. This gelatinized membrane may become more and more liquefied until large numbers of cells are buried up in a common jelly-like mass, and it is in this way that the various forms of zoogloea arise. The majority of bacteria have no such envelope at all, or at most a thin invisible mucilaginous coat. The formation is often largely dependent upon the nature of the food-stuffs available (Fig. 7, b and c\ Inasmuch as many bacteria secrete large quantities of mucilage without forming a definite envelope, the word ' capsule ' should be sparingly used and restricted to clearly marked sheaths around the cell. Only in such cases has it any classificatory value (Leuconostoc]. Moreover, the clear spaces visible around bacteria in dried preparations are frequently artificial products. These are par- ticularly prone to arise in fluids like blood and lymph, which con- tain albuminous substances, the albumen being seemingly precipi- tated upon the bacteria, around which it forms a coating that easily takes up stain. When dry- ing takes place the cells, being the last to give up their moisture, shrink away from this envelope and each one appears to be sur- rounded by a capsule (Fig. 7, a). That this is, however, nothing but an artificial structure is shown by the fact that the same bacteria which in the blood of an animal exhibit a capsule are, with some doubtful exceptions, both in the tissues and in cultures, quite devoid of one. In a mouse which has died of anthrax, the bacteria in the blood seem to have a capsule (Fig. 7, a], whilst those in sections of the kidney are totally devoid of one. That this is not due to a difference of the modes of growth in the blood and in the tissues is shown by the fact that the naked anthrax bacilli from an agar culture become provided with an apparent * capsule ' if they are rubbed up with a drop of blood from a healthy mouse and allowed to dry upon a cover-glass. Among the trichobacteria (Cladothrix^ CrcnotJirix] and some of the cyanophyceae (Tolypothrix, Lyngbya), the outer layers of the cell-wall undergo a hardening process which leads to the production of a firm sheath FIG. 7. Capsules and mucous sheaths, a, Bac. an- t/tracis\\\\h so-called capsules in adi ied streak-preparation from the liver of a mouse ; for the nature of these and of the capsules of other pathogenic bacteria, see p. 10. b—d, Lenconosloc mesenteroides (frog spawn fungus) ; 6, on non-saccharine media, without sheath ; c, with mucous sheath on medium containing sugar (b-c from Liesenberg and Zopf) ; d, older zoogloea mass with chains of cells (from van Tieghem). Magn. a 1500, b and c 1200, ^500. l-'L\ER STRUCli'Ri: O/-' Tilt li.lt it: RIAL (ELL u enclosing the cylindrical cells in such a way that they arc freely movable within (Figs. 2. and ",). At the period of reproduction the cells, them- selves devoid of sheaths, become furnished with cilia, and, escaping from the open end of the sheath, swim away and give rise to fresh colonies. In this way whole forests of cladothrix are emptied of their cells and there remain only the stiff sheaths which finally swell up and become disintegrated. Occasionally the sheaths become fossilized as it were, by the deposition upon them of oxide of iron. They are then very resistant to decay, and accumulate sometimes in enormous quantities in chalybeate springs and ferruginous marsh water (see Chap. VII). The term 'sheath' can only be applied when a distinct tube is formed in which the chain of cells are enclosed ; colourless spaces in stained preparations of filamentous bacteria do not necessarily imply the existence of a sheath, being often the result of plasmolysis. CHAPTER II MORPHOLOGY— continued Pigments, Intracellular Products ; Movement and Organs of Locomo- tion ; Cell-division ; Spore-formation and Germination. MOST bacteria are individually colourless, and appear, even when massed together in a pure culture, cither white or of a yellowish tinge. There are, however, a considerable number of species, the chromogcnic or pigment-bacteria, remarkable for the brilliant colouration of their cultures. Some of the Sarcinac, for instance, have a bright yellow tint, StapJiylococciis pyogcnns anrci'.s is golden yellow or orange, B. brunncus yellowish brown, Micrococcns agilis. Bacillus prodigiosus^ and Spirillum rubrum each a different shade of red, B. cyanogenus (the bacillus of blue milk) blue, B, violaceus a deep violet ; and many kinds of water-bacteria, as well as the bacillus of blue pus, give origin to brightly fluorescent pigments. The production of all these colouring matters is very variable and largely dependent upon the conditions of growth, upon the composition and reaction of the culture media, and upon the influence of oxygen, light, and heat. Most pigment-bacteria appear under the microscope to be colourless, and the question at once suggests itself whether the colouring matter is really contained within the cell. In B. prodigiosus, the cause of the ' bleeding host,' the pigment can be seen in the form of granules scattered about between the colourless bacteria, so that in this case there can be no doubt as to the seat of the colour of the cultures. The various fluorescent substances also are secretions of the bacteria with which they are associated, and they diffuse into the culture medium, which often fluoresces brightly throughout its whole thickness. The pigment of B. cyanogenus also is dissolved in the culture medium. In most pigment-bacteria similar con- ditions prevail, that is to say, they are ' chromoparous ' (6). Some species, on the other hand, are actually ' chromophorousl i. e. the protoplasm itself is coloured. This is the case with the sulphur bacteria Chromatium and Thiocystis, and with some sap-green species (B. wrens). As regards these last, however, it is undecided whether they are bacteria or minute algae (7). BACTERIAL PI CM EX TS AND CELL-CONTENTS 13 Finally, there are bacteria, Z>. I'iolaccus for example, in which the pigment is lodged mainly in the cell-wall ; such forms may be termed ' parachromatO' phorotts.1 There is conclusive evidence that the red colouring matter of the sulphur bacteria, ' bacteriopurpurin,' performs the same functions as the chlorophyll of higher plants, assimilating carbonic dioxide under the in- fluence of light and giving off free oxygen (see Chap. VII). Only the outer layer of the cell contents is charged with pigment, the central mass being colourless. In all probability the colouring matter is of importance physiologically only where it is bound up with the protoplasm. In all the ' chromoparous ' bacteria the pigment is an excretion merely, and, as might have been expected, chemical and spectroscopic analysis fail to show that it has any connexion with assimilation. Some of the bacterial pigments are of the nature of lipochromes and are related to the fats ; others have a basic character and resemble the ptomaines. Many seem to belong to the proteins, and the colouring matter of B. cyaneo-fnscus has a composition similar to that of indigo. Differentiated cell-contents, such as the starch or aleurone grains of higher plants, are wanting in most bacteria. The protoplasm stains a uniform golden yellow when treated with iodine. Some of the butyric acid bacteria, however, as well as various species inhabiting the human mouth, form an exception to this rule, and take on a blue or deep violet tint. Very little is known about the substance to which this reaction (granulose reaction) is due ; it has been called granulose merely on the strength of its behaviour towards iodine. Whether its chemical composition is similar to that of the granulose of starch cannot be decided by this reaction alone. It seems to be a carbohydrate, however, and carbohydrates as food are necessary for its growth. The mouth bacteria obtain these of course from our food, and the butyric acid organisms from the fermenting substances in which they grow. The ' granulose ' makes its appearance in the bacterial cell in the form of very minute grains, so that after treatment with iodine the yellow protoplasm seems full of black points. These granules become considerably larger, and then seem to diffuse through the cell contents, so that finally the whole cell stains blue or violet. In the butyric acid bacteria granulose is at first absent, but is formed when the time of sporulation draws near. In that part of the bacterium, however, where the spore appears, none is deposited, the protoplasm staining from first to last yellow with iodine. It would seem that we have here an instance of division of labour, the swollen end of the bacterial cell being devoted to sporulation, and the cylindrical portion serving as a manufactory and storehouse for granulose, from which the spore may be nourished (Fig. u, c—f). i4 MORPHOLOGY Absolutely unique in the whole of organic creation is the case of those bacteria whose protoplasm contains free sulphur (Chap. VII). In these ' sulphur-bacteria ' the cell is often crammed full of highly refracting globules, which are soluble in alcohol, xylol, bisulphide of carbon, and alkalies, and give other reactions of pure sulphur. It is in a non-crystalline, soft, amorphous state, and when dissolved out by carbon disulphide leaves delicate clear vacuoles in the protoplasm. Deposits of other substances within the bacterial cell have not as yet been observed, with the exception of fat or oil, which sometimes appear in the form of minute globules, particularly in old cultures. Movements and Organs of Locomotion. All bacteria, whether spontaneously mobile or not, are, when suspended in water, in constant motion. Examined under a high power, they are seen to be shaken by a rapid oscillatory movement. This, the so-called Brownian movement (8), is in no sense a vital phenomenon, but is common to all finely- divided solid particles suspended in liquids. It may be observed in the particles of gamboge or inclian ink rubbed up with a little water. Its cause is not well understood. As a general rule there is no danger of mistaking the trembling motion for the spontaneous movements of bacteria. There are two kinds of independent motion distinguishable among bac- teria— the ordinary swimming movement, and the rarer ' oscillation ' peculiar to some trichobactcria and similar to the movements of some cyanophyceae. Among the cocci, Micrococcns agilis is the only one endowed with the loco- motory powers, but of the bacilli a large number are actively mobile, such as B. typhi, the butyric bacteria, and the majority of the putrefactive organisms. The vibriones and spirilla, too, are good swimmers. The bacilli of tuber- culosis, diphtheria, anthrax, many of the pigment bacteria, and the acetic and lactic ferments are permanently immobile. When bacteria in active movement are examined under a high power they seem to shoot across the field of view with amazing speed ; but this velocity is only apparent, being due to the magnification of the distances they cover. Reduced to actual figures, the absolute rate of progress is 10 cm. in 15 minutes, or about ^ mm. per second — in proportion to their size a very fair speed. Locomotion is effected by special organs, the cilia or flagella. In the fresh state, or in bacteria stained in the ordinary way with anilin colours, the cilia are not visible, and special methods of preparation are necessary to render them so. That worked out by Loeffler (9) has the claim to priority and is the best. He employs in the first place a mordant composed of an iron salt and tannin, which causes the stain to be thrown down not only within the cell, but also upon its surface and upon the surface of the cilia, which LOCOMOTION ANH LOCOMOTORY ORGANS then appear deeply coloured. Both bacteria and their cilia arc by this process made to look thicker than they really arc, and appear therefore more clearly. According to the arrangement of the cilia upon the cell the bacteria may be divided into three groups — monotriclia, lopliotricJia, and peritricha(\$). The members of the first division bear a single flagellum at one end of the cell (Figs. 8, a, and 23) ; examples are the cholera germ and other vibrios, and />. pyocyancns. The lophotrichous bacteria have in place of the single flagellum a brush or tuft of cilia (Spirillum, many putrefactive bacteria ; Figs. 8, b ; 22, a; 12) and the peritrichous forms have their whole surface beset with cilia more or less thickly arranged, so that they appear almost as though they had a shaggy coat (B. typlii, B. coli cominunis, some butyric ferments, B. snbtilis, B. pro- tens, and many others ; Figs. 8, c, e, and ii, 13, 22, 24, 28). The arrangement of the cilia is constant in each species, and even the number of cilia united to form the terminal tuft has a taxo- nomic value. Functionally and structurally the cilia of bacteria correspond to the analogous organs in other groups, such as the swarm -spores of algae and fungi, the infusoria, and the ciliated cells of metazoa. They consist of long delicate threads of protoplasmatic sub- stance, which vibrate to and fro and propel the bacterium through the water as a boat is propelled by oars. They grow out slowly from the body of the cell (Fig. 8, , !opliGtrichous(>S//r/////>« nn- dula); c, peritrichous (typhoid bacilli) ; d, develop- ment during fission of the cilia tuft of Spirillum undula\ e, partial and (to the right) complete looping of the cilia in Rctc, subtilis. Magn. a-e 2250. In Figs, a, b, c, the structure of the cell-con- tents has been taken from iodine preparations (Fig. 5) in order to illustrate the structure of bac- teria as far as is known at the present time. In Figs, (/and e the contents are schematically shaded, for in these preparations (stained by Loeifler's method) the precipitation of colour on the cell- surface conceals the structure. See also Figs, n, 12, 13, 17, 22, 23, 24, 26 and 28, which give fun her examples of various ciliation. 16 MORPHOLOGY peritrichous bacteria then appear to be surrounded by a mass of bubbles (Fig. 8, 4 The speed of movement of the bacteria is subject to great variations, the cilia being often paralyzed by noxious physiological conditions. Lack of oxygen, insufficient nutriment, and the accumulation of acid in old cultures are all factors which bring about this paralysis, and it is only necessary to remove them (by neutralization of the acid, by removing the cover-glass, by the addition of sugar, asparagin, &c.) to at once restore movement to the cilia. That bacteria are motionless is not therefore a proof that they belong to a non-motile species, and in some cases much experience is necessary for decision. The linear progression of bacteria through a liquid is always accompanied by a rotation around the longer axis of the cell, as is the case with the swarm-spores of the lower thallophytes. In the flagellate infusoria the end of the cell bearing the flagellum is always in advance in swimming, and probably the lophotrichous and monotrichous bacteria move in the same way, a reversal of the path necessitating a revolution of 1 80° around the transverse axis of the cell. The peritrichous bacteria have a similar mode of progression to that of the members of the other groups, but in addition they often exhibit curious tumbling movements, the cell hurrying across the field of view and turning somersaults the whole way. In Bcggiatoa, one of the filamentous bacteria, remarkable slow oscilla- tory movements of the threads are observed, isolated fragments of which are also able to glide to and fro as diatoms do. Both these types of movement occur too among the cyanophyceae, giving the name to one group of forms, the Oscillariae. The cause of the phenomena is quite incomprehensible, no organs of locomotion having been discovered, and the cell-wall being apparently closed all round, so that the protoplasm cannot be perceptibly extruded anywhere. It is, however, highly improbable that the motion is independent of the protoplasm. More delicate methods of research will perhaps enable us to clear up the mystery. The term flexile has been applied to filaments which, although per se rigid and stiff, are not straight, but twisted and thrown into curves. It is supposed that such flexile filaments have somewhat less rigid cell-walls, which yield to the strain exerted on them by the cell contents. These flexile threads occur in all trichobacteria, but in the haplobacteria the membrane is always rigid. Kinks and twists in filaments seem to arise frequently by the mechanical displacement of neighbouring cells. The subject of flexility needs further investigation. Reproduction of Bacteria by Fission (11). Given suitable conditions of nourishment, bacterial cells, like those of all other organisms, divide into two when they reach a certain size. In REPRODUCTION OF BACTERIA HY FISSION <7 the filaments of the trichobacteria cell-division means growth and increase of length of the filament, and the process can only be called multiplication \vhcn a cell detaches itself from its fellows and grows out into a new filament. Among the unicellular haplobacteria, however, each cell-division means duplication of the individual. The procedure follows the same course as it docs in the tissues of the higher plants. The cell first increases in length, and then becomes divided into two by a transverse wall. The spherical bacteria (cocci) assume an ellipsoidal figure before dividing, and the two new cells are at first flattened where they arc in contact, rounding off to perfect spheres again as they separate. Of the finer details of the process nothing has been seen among the bacteria, nor anything resembling those changes in the arrangement of the cell-contents which characterize cell-division in the higher organisms. The protoplasm is simply abstricted into two parts separated by the in- growing cell-wall, as in CladopJwra. In the filaments of this alga cell- division is ushered in by the deposi- tion of a ring of cellulose on the inner surface of the cell-wall, where the new partition is to arise (Fig. 9, a), By constant addition to its inner edge this ring grows broader, until at last, cutting through the proto- plasm, it stretches right across the lumen of the cell and divides it into a. FIG. 9. Transverse division of a multinuclear living cell of an alga ( Cladoptwra fracta). The new cell- wall (as in all multinuclear cells) arises independently of the division of the nuclei. In Fig. a the new trans- verse cell-membrane grows out as a ring at right angles to the sides of tlie cell and appears (in optical section) as rod-like outgrowths from the latter, the free ends being surrounded by granular piotoplasm. The large round bodies are starch grains. Fig. b represents an older stage, the new membrane is com- plete with the exception of a small spot in the centre. The figure is meant to give an idea of what probably takes place during the fission of bacteria which an: too minute to allow the process to be followed. From Strasburger. Magn. 600. two equal parts. There seems little doubt that division of the bacterial cell takes place in the same way, but the details of the process are too minute even for our best microscopes to follow. Under the most favourable conditions of temperature and nutrition cell- division takes place in a very short time. B. snbtilis, the ' hay-bacillus,' completes the process in half an hour. The cholera vibrio needs only twenty minutes, so that in one day a single 'comma-bacillus' would rejoice in a progeny of sixteen Jnindrcd trillions. This mass of bacteria would contain one Jnindrcd tons of solid residue, and it would thus be necessary to make experiments on a gigantic scale to allow even one single cell to multiply with perfect freedom. In nature, of course, this increase in geometrical progression can never take place: in the first place, because the necessary supply of food is never present, not even in the diseased body, and then again because many of the bacteria soon perish, and the accumulation FISCHEK i8 MORPHOLOGY of the excreta (bacterial products such as acids, &c.), particularly in pure cultures, arrests development. As standards of comparison it may be mentioned that a complete division of nucleus and cell in the staminal hairs in Tradescantia takes from eighty to one hundred minutes, and that the process may be effected in Amoeba in from ten to twenty minutes. The rate of multiplication among the bacteria is therefore nothing extra- ordinary, particularly when we consider the simple structure of their cells and the absence of a complicated karyokinetic process. When a cylindrical bacterium divides into two it is immaterial, as far as the result is concerned, whether the division is longitudinal or transverse ; but, as a matter of fact, we find that it is always transverse, be the cylinder straight or spirally twisted. This is in harmony with the generally observed law in cell-division, that the new membrane is always formed in the most economical manner ; that is to say, in such a way that a minimum of material is required. In cylindrical cells this is manifestly the transversal position. When the cells resulting from such division remain connected, chains and filaments arise, particularly in the case of non-motile species. They occur sometimes even among motile forms, such as the cholera vibrio (Fig. 28, /£), although in this case the moving cells are more easily separated. Inasmuch as transverse fission is the only mode of increase among cylindrical bacteria, it follows that chains or filaments are the only kinds of colony or ' growth-form ' that can occur, save where there is a subsequent shifting of single cells (as in Cladothrix). In the monotrichous and lophotrichous bacteria it is always the non- ciliated end of the dividing cell which bears the cilia for the new individual (Fig. 8, d), the inner ends of the two rods never bearing them. In all cases where the cell seems to have cilia at both poles there are in reality two young cells still united by their inner ends. A curious possibility arises out of this fact. At every division it is only one individual that is provided with new cilia, the other cell bearing the old ones, and this process may be repeated time after time. As a result, it may happen that of two bacteria swimming about and not as yet disconnected one has brand-new cilia, and the other a set which has lived through hundreds of generations. In peritrichous forms it seems probable that, during the elongation of the cell preparatory to fission, new cilia arise between the old ones along the side of the cell. Among the spherical bacteria it is manifestly, as far as the economy of the cell is concerned, a matter of indifference in which direction the plane of fission lies, because every plane that passes through the centre of the cell represents a minimum. A predilection for one particular plane must there- fore be the outward expression of hereditary morphological characters that possess a classificatory value. The closest resemblance to the cylindrical bacteria is shown by those spherical forms in which the planes of fission of REPRODUCTION OF BACTERIA RY FISSION 19 successive generations are all parallel. In this case, if the cells remain attached to each other, unbranched chains arise as in Streptococcus Pyogcnes (Fig. 10, #), a pus bacterium, or as in Leuconostoc mesenteroides (Fig. 7, .\ vibrio, nor spirillum forms appear. The various twistings and distortions of filaments, and aggregations of motionless gonidia, which have been described as phases of plcomorphic development, are quite fortuitous. Pus-cocci (Stapkylococci] may be cultivated in any number of different media, but they appear with unalterable persistence in the form of little spheres (Fig. 28, a). The cholera vibrio occurs in the form of a slightly curved rod, and it occurs in this form only. The only observable variation in growth is that in some cultures isolated cells may prevail, and in others chains (Fig. 28, /•). In a hay-infusion culture of B. subtilis, for instance, we meet side by side motile and motionless single cells (Fig. 13, a, b), and, especially in the surface pellicles, motile and motionless chains (Fig. 13, b}. In all these the individual bacterium remains unchanged, a unicellular, peritrichous-ciliate, actively motile rod (Fig. 13, a). The motionless rods arise from any causes which bring about temporary paralysis of the cilia, and the motile chains from the clinging together of successive genera- tions of cells. In a fresh culture the fluid is uniformly turbid, with actively moving isolated rods only. These are afterwards driven by lack of oxygen to the surface of the culture, where they grow out into long, motionless, spore-bearing chains devoid of cilia, the aggre- gation of which forms a membrane or pellicle on the surface of the liquid. This completes the cycle of forms. These instances will suffice to show that pleomorphism in its true sense does not exist. In all the simple bacteria (Jiaplo-bactcria) the 'growth-form ' only changes. Single cells unite to chains, clusters, or zoogloea, according to the substratum they live in, but the form of the individual of the vegetative cell remains the same. That proper and improper nutriment must have a great effect upon the size of the cell is evident. Giant and dwarf forms occur among the bacteria as among all other organisms, and have the same significance. But for all species of bacteria there is an average size and form, deviations from which are not more extensive than they are among other organisms : always assuming that the conditions of existence are suitable, that the bacteria are in a healthy condition, which is far less frequently the case than we are apt to think. Imagine a few thousand animals cooped up together in a confined space, plentifully supplied with food, but without provision for the removal of their dejecta. A few hours would suffice to bring about a frightful state of things. And these are just the conditions under which FIG. 12. Cladothrix dichotoma, formation of swarm spores. The sheath (stippled) of the left branch has opened and gives exit to a swarm spore; in the right branch a whole group of cells has changed into swarm spores, each with a lateral tuft of cilia. On this side too the sheath is swollen up, loosened. Magn. 1000. 26 TAXUNOM Y the bacteria are living in agar cultures, in all our culture media in fact. In such circumstances it is no wonder if some of the cells grow into mis- shapen, feeble involution forms, with their physiological functions, such as virulence or fermentative power, weakened or suppressed (16). Bacteria, like all other living things, produce crippled and deformed individuals when forced to live under unsuitable conditions (Fig. 14). These abnormal bacterial cells are known as ' involution forms.' The causes of involution are of various kinds. Acetic bacteria, for instance (Fig. 14, c-d), produce monstrosities if their own fermentation product (acetic acid) accu- mulates beyond a certain point, and also if the temperature exceeds the optimum. In B. snbtilis involution forms arise if the relative propor- tions of nitrogen and carbon in the culture medium are not suitable — a solution of o-i per cent, asparagin and 10 per cent, sugar, for instance. In other cases a high percentage of neutral salts induces them. A curious case of involution is that shown by the bacteria in the root tubercles of the leguminosae (see Chap. X). The shapes that the cells assume arc very varied. Irregular, swollen, or spindle-shaped rods and twisted chains are common, and the protru- sion of short lateral processes from the cells often gives the growths the appearance of a branched system (Fig. 14). At the same time the cell-contents are reduced and stain badly, a few granules being appa- rently all that is left in the cell. Cells which show a high degree of involution are dead, and cannot, even in the most favourable conditions, be revived. They are common in old cultures, and particularly so in those of strictly parasitic forms, such as the diphtheria and tubercle bacilli, that even in the most nourishing media do not find quite the conditions they require. Such branched involution forms have been thought by some to show that the bacteria of diphtheria and tubercle (Fig. 14, /i, g] are fungi of a higher order of growth than their ordinary rod form would indicate. It has been suggested that the rods are merely a stage in the development of a true filamentous bacterium or even a hyphomycete, and new names have accordingly been given to the organisms, the tubercle bacteria being placed ... •'•"" ..." .,..<*^- •-:•'" - FIG. 13. Bacillus subtilis in hay infusion. The figure shows the complete cycle of forms, a, Peritri- chous motile short rod ; b, non-motile rods and chains ; d, motile chains ; c, spores in non-motile rods and chains that unite on the surface of the infusion to form a thick whitish pellicle e. Magn. a-d 1500, e (from Brefeld) 250. INVOLUTION: ATTENUATION 27 in a new genus Mycobactcriitin or Tnbcrciiloinyccs, and the diphtheria para- site in another, Coryncbactcrimn. In the writer's opinion such an alteration is not justified, for the branched forms of tubercle bacillus that arise in three to six months old colonies arc by no means common, and agree closely in appearance with the involution forms in the root-nodules of leguminosae Fig. 14, e-f, and of the acetic bacteria 'Fig. 14, c-d. If the influences which give rise to involution forms be restrained within certain limits, a general weakening or attenuation of the bacteria is !•" Hi. 14. Involution forms, a, Bac. siibtilis from a four-days old culture containing i % sal ammoniac, 2 /£ grape sugar, and 0-5 % nutritive salts (weakly acid), b, Typhoid-like bacilli from water in hay infusion with 4 % sal ammoniac; they are non-motile, without cilia, and suggestive of the bacteria of root-nodules (e and_/\ c, Bac- terium aceti at 3Q°-4l (from E. Chr. Hansen). d, Bacterium Fasteurianum, 7 hours at 34° (from Hansen). e, Bac- teroids from the root-nodules of Vicia villosa \ the round spots are the still retainable remnants of cell-contents (from Morck). f, Bacteroids from Lupinus albtis (from Morck ; the four-armed one is from Vicia villosa). r, Tubercle bacillus, branched filament from sputum (from Coppen Jones). //, Diphtheria bacilli, so-called branched forms ; they are certainly involution forms (from Bernheim and Folger). Magn. a and b 1500, c and d 100, e and /about 1500,^ 1250, It about 100. effected. This condition arises spontaneously in long-continued cultivation on artificial media ; virulence, fermentative power, and other functions being diminished. They may be revived again, however, if the process has stopped short of involution, by restoring the bacteria to a more natural environment: in the pathogenic species 'by repeated passage through the animal body, in the ferment organisms by giving them renewed oppor- tunity for active fermentation. The attenuation induced by prolonged artificial culture can be secured in a much shorter time by exposure to more noxious agencies. The most important form of enfceblement, the loss of infectious power, can be induced 28 TAXONOMY by almost all operations injurious to life, if only the proper degree of strength be chosen. Anthrax bacilli are attenuated by a few hours' exposure to direct sunlight or by the addition to the culture media of o-i- 0-2 per cent, phenol. Trichloride of iodine is effectual in the case of the diphtheria and tetanus bacilli. By careful exposure to high temperatures, Pasteur was able to produce a less virulent breed of anthrax bacilli which retained their acquired characters for a long time (17). The change effected was the same whether the cultures were maintained at 42-5° C. for twenty- eight days, at 43° for six days, at 47° for four hours, or at 52° for fifteen minutes. The temperature optimum for Bac. antJiracis is between 30° and 37°, the maximum about 42° or 43°, and the lethal limit lies between 50° and 60°, so that it is evident that attenuation is effected the more quickly the nearer the temperature approaches the lethal limit. That the decrease of virulence is only one phase of a general weakening of all the functions is shown by the fact that the bacteria in attenuated cul- tures have lost the power of forming spores : have become asporogenousQfy. At first sight this seems to be a result of the greatest importance. Spore formation is one of the principal morphological features of the cell. If we were really able to suppress it, and to suppress it so successfully that it never made its reappearance even in the most vigorous cultures, we should indeed have done a great thing, for we should have obtained nothing less than the long desired proof of the hereditary transmission of acquired charac- ters. But appearances, alas, are deceptive. It is no more possible to obtain in this way a breed of sporeless anthrax bacilli than it is to rear a tailless race of mice by incessantly cutting off their tails. All we do is to hinder the production of fully ripe, resistant spores : rudimentary spores are formed. We attain nothing more than a general enfeeblement of the cell affecting all its functions equally. This is proved by the fact that such ' attenuated ' and ' sporeless ' cultures get weaker and weaker and finally die. By the inoculation of the weakened bacteria into suitable animals and several passages through the body, in short, by a bracing treat- ment, virulence is restored and resistant, fully ripe spores appear again. The micro-organisms behave, in fact, just as sickly plants do when removed to healthy surroundings. The importance of the experimental modification of virulence for the attainment of artificial immunity will be considered in a later chapter. All these considerations show us that external agencies (in the brief period which our experiments allow) are unable to bring about a lasting, hereditary change in the structure of bacteria. The morphological characters remain constant. The variety always throws back to the typical form, the concep- tion of species and genera having the same value among bacteria as among the higher organisms. Billroth's views, according to which all the bacteria occurring in an infected wound were different developmental stages of the PLEOGENY 29 same species (Ci>cci>l>tn-tiTia sif/tid), can be no longer held. Like Zopf's far-reaching speculations on the cycle of forms among bacteria, they are matters of history, and they are nothing more. The similar ideas of Nageli have been unable to hold out in the face of more recent dis- coveries, and there can be no doubt that the justification of Conn's original conception of the possible systematic classification of the bacteria is now generally recognized. Less simple to decide than the question of pleomorphism is the question of physiological change or plcogeny*. All bacteria possess to a certain extent the power of living on different kinds of substrata, and the com- position of these determines in a great measure the nature of the chemical changes which the bacteria set up. According to the degree to which this power is developed, we may conveniently divide the bacteria into two classes, the monotropliic and the polytrophic. Among the members of the first group the requirements of nutrition are specific and clearly defined. The composition of the substrata may vary only within very narrow limits, and, as a result, the metabolic products also of these bacteria and the func- tions they are able to perform are specific and well defined. As typical examples of the monotrophic group may be mentioned the sulphur bacteria, the nitrifying organisms, all the truly parasitic bacteria, and those found in the root-nodules of the leguminosae. Among the great host of ferments and putrefactive organisms, too, there are monotrophic forms with specific properties. Such are the bacteria of the acetic, lactic, and some butyric fermentations, and the bacteria which decompose urine (urea ferment). Some of the butyric bacteria, on the other hand, are polytrophic, and, besides possessing specific fermentative powers, arc able to break up albuminous compounds ; they are saprogenic as well as zymogenic. Others of the same class are pathogenic also, and can live in the tissues of the animal body (malignant oedema, quarter evil). We find, too, forms like B. vulgaris with pronounced saprogenic (putrefactive) powers, which are yet able to grow on non-putrefactive substances and cause them to ferment. Again, many saprophytic bacteria are unable to exist in the animal body, while others (typhoid, cholera) are decidedly polytrophic in this respect, and multiply rapidly in the tissues. It is not necessary to give parallel examples of differences among other organisms. As might be expected, it is in monotrophic forms that the ' specific characters,' both morphological and functional, are most sharply marked. But they undergo no lasting change even in those bacteria which are physio- logically the most versatile ; and the attainment in our cultures of new varieties, varieties with acquired characters that are permanent and heredi- tary, is not to be expected. We are able, it is true, to obtain cultures of * Detailed instances will be found in Chaps. V, XI, XII, XIII, and XV. 30 TAXONOMY pathogenic species with every desirable gradation of virulence, but they are not new varieties. They are mere laboratory stocks, which revert at once to the original type as soon as the original conditions of life are restored *. It has not been hitherto possible to entirely suppress a single biological character in any species. The supposed conversion of the anthrax bacillus into the harmless B. sribtilis was never realized, and, like most reports of a similar character, arose from imperfection in the technique of pure culture. Although there is no longer any doubt that the various kinds of bacteria constitute definite species and genera, it would be futile to minimise the great difficulty that confronts us as soon as we try to arrange them in a system of classification. The morphological similarity of the spherical forms (cocci) and the close resemblance to each other of the rod- shaped bacilli make a classification based on purely morphological data quite impossible. Physiological characters have therefore been made use of as auxiliaries — for instance : growth on various media and need for oxygen or particular forms of nourishment ; specific products such as pigments, granulose, sulphur, fluorescence, phosphorescence ; specific chemical changes such as fermentation, putrefaction, disease. A reliable description of the species of bacteria is a task for the future, and will only be attained by the united labours of pathologists, physiologists, chemists, and botanists (19). Much has been done by arranging them in groups according to their most prominent physiological characters. Examples of such groups are the saprogenic or putrefactive, the zymogenic or fermentative, the chromogenic or pigment-producing, the phosphorescent, the thermogenic, the nitrifying, the iron bacteria, and the sulphur and purple bacteria. Such a classification is not without value, but it is not justifiable to make generic names on this principle, and use them as though the groups they indicate were equivalent to true morphological genera. Such names as Photobacteriuni, Nitro- bactert Nitrosomonas, Grannlobacter (for butyric bacteria with the granulose reaction), Halibacterinui (for marine forms), Gouococcus, and Proteus (for several putrefactive bacteria) are names that should not be admitted in a systematic classification. As working or trivial names they are useful enough, but have no claim to equality with genera based on morphological characters, which must, after all, form the ground-work of all systems of classification. An attempt must be made, despite the dearth of material, to obtain clear morphological definitions of the genera at least, even if it be impossible at present for species. This was Cohn's guiding principle (20), and must be adhered to. The very factor to which the progress of bacteriology is mainly due, namely the number and variety of its students, has been a hindrance to the proper growth of a classification. Side by The various ' races' of brewery yeasts have a different value to these ' attenuated' bacteria. CLA SSIFICA TION 3 r side with the medically trained ' bacteriologist,' to whom the principles and meaning of classification were unfamiliar, there have been investigators from every other branch of biology — brewers, agriculturists, and even chemists, all vying with each other in the manufacture of species. It is not argued for a moment that botanists alone have the right to set up a system; but it cannot be too often repeated that other investigators, if their taxonomic schemes are to be of any value, must proceed on the recognized and established principles of classification. As things are, there is no agreement even as to the value to be attached to the few clearly marked morphological features which do exist. The only point of unanimity is the adoption of Cohn's groups founded on the shape of the cell (coccus, rod, spiral, or filament). The importance of the distinction between fila- mentous bacteria and those forms which consist of a single cell deserves more emphasis than it usually receives. The filamentous bacteria, tricJio- bacieria, constitute an order, separate from the haplobacteria^ in which the colonies of cells are mere ' growth-forms ' or social aggregates of individuals, such as, for example, the packets of Sarcinae, or the cobweb-like zoogloca of B. "culgaris. The power of movement or its absence has very properly been regarded as a distinctive feature, but the constancy of the ciliation (mono-, lopho-, peritrichous) has been undervalued. A cholera vibrio or a bacillus pyocyaneus has only one flagellum (as a rare exception two) ; the typhoid bacillus, B. siibiilis, and many others are always peritrichous. Lophotrichous forms like the spirilla and other aquatic bacteria, or the bacillus of blue milk (B. syncyaneiis), always have a polar tuft of cilia, the number of which is approximately known and fairly constant. That their numbers in a stained preparation are often irregular is due to the delicacy of the structures, which are easily thrown off, and not to any original irregularity. In the motile bacteria, as in the flagellate infusoria, the number and arrangement of the cilia are morphological characters of fundamental systematic value. Another is the form of the sporulating rod, which, although described by some as variable, possesses in reality the constancy required of systematic characters. The anthrax bacillus retains its cylindrical shape, the tetanus bacillus becomes invariably swollen at one end, like a drum-stick (plcctron},a.i\d some of the butyric bacteria assume the shape of a spindle at the time of spore- formation. Exceptions to the rule are generally deformed individuals, and are very rare, not one in a hundred. A careful discrimination of these two characters, ciliation and sporulation, enables us to arrange the chaotic multi- tude of rod-shaped bacteria into a few well-defined genera and sub-genera. It may be argued that in many cases the spores are unknown. This is true, but it is no reason why we should not classify the better known forms into ' good ' genera, relegating the others provisionally, according to the shape of their cilia, to genera where the sporulation is unaccompanied by change 32 TAXONOMY of shape in the rod. The names of the genera might be formed con- veniently in such a way that the root of the word indicated the shape of the cell, and the termination the arrangement of the cilia. The root-words might be baktron (rod), blaster (spindle), and plcctron (drum-stick), and the terminations -inium for monotrichous, -ilium for lophotrichous, and -idium for peritrichous types. The classification of the less numerous spirilla is, as the table shows, a simpler matter. Among the Coccaceae the manner of cell-division has already been employed as a generic character, but greater weight should be attached to the contrast between two more comprehensive groups into which they naturally fall. These are the Homoccocaccae, where the planes of fission follow a definite sequence, and the Allococcaceae, in which such regularity is not found (see pp. 18 and 19). The different meanings attached to the words Bacillus and Bacterium. deserve notice. In two of the most recent systems of classification (21) the senses in which they are used have little in common. Lehmann and Neumann apply the word Bacterium to all rod-shaped forms in which spores are unknown (a point in regard to which any day may bring a change in our knowledge), the genus Bacillus embracing those in which spores have been found. Of the manner of ciliation no notice is taken. Migula, on the other hand, uses the term Bacterium for all non-motile rods, and Bacillus for peritrichous species, the remaining motile forms, both lophotrichous and monotrichous, constituting a new genus, Pscudomouas. This classification very properly regards the presence or absence of spores as unimportant ; but the shape of the spore-bearing rod is neglected, and no distinction is made between monotrichous and lophotrichous species. Perhaps the best plan would be to drop altogether the word Bacterium as a generic term, seeing that it is now used as a collective name for the whole group of micro- organisms. The word Bacillus might then be used, in memory of Koch's first work, for all those species which, like the anthrax parasite, are non- motile and retain their shape during sporulation. At present we are not justified in regarding the presence or absence of capsules as sufficiently important to constitute a generic character, although very useful for the differentiation of species. The trichobacteria are so few in number that it will suffice at present to unite them in a single family representative of another order. Based on the characters that have been under consideration, a classifi- cation of the bacteria might be arranged as follows :— Order i. HAPLOBACTERINAE. Vegetative phase unicellular, spherical, cylindrical, or spirally twisted ; isolated or united in chains or clusters. Family i. COCCACEAE. Vegetative cell spherical. CLASSlFlCATiax 33 Sub-family i. AUococcaceae. Planes of fission without definite sequence ; no pronounced colonies or growth- forms, cells isolated or in short chains or irregular clusters. Genus MiCROCOCCUS, Cohn. Non-motile. Includes most cocci, the pathological ' staphylococci,' &c. Genus PLANOCOCCUS, Migula. Motile. Sub-family 2. Homococcaceae. Planes of fission in definite sequence. Genus SARCINA, Goodsir. Three planes of division at right angles to each other. Cubical colonies, non-motile. Genus PLANOSARCINA, Migula. Similar to Sarcina, but monotrichous, ciliate, and motile. Genus PEDIOCOCCUS, Lindner. Two planes of fission, alternate and at right angles. Micrococcus ietragemis, Thiopedia (a sulphur organism), and probably some species usually termed micrococcus. Genus STREPTOCOCCUS, Billroth. Planes of fission parallel, giving rise to chains ; the pathological Streptococci and Leuconostoc. Family 2. BACILLACEAE. Vegetative cell straight, cylindrical, ellipsoidal or egg-shaped ; very short forms difficult to distinguish from cocci. Fission always transverse. Sub-family I. Bacilleae. Spore-forming rods cylindrical, unchanged. Genus BACILLUS, Cohn. Non-motile. J3. anthracis, B. diphtheriac, &c. Genus BACTRINIUM, A. Fischer. Motile, monotrichous, with terminal cilium : includes provisionally all monotrichous rods whose spores are as yet unknown, e. g. Bac. pyocyaneiis, Genus BACTRILLUM, A. Fischer. Motile, with lophotrichous ciliation. Includes provisionally Bac, cyanogemis, and many other sporeless forms. Genus BACTRIDIUM, A. Fischer. Motile, peritrichous, in some spores as yet un- known. Very numerous representatives, e. g. B. subtilis, B. megatherium, B. intlgaris (old genus Proteus), B. typhi, and B. coli. * Sub-family 2. Clostridieae. Rods spindle-shaped during sporulation. Genus CLOSTRIDIUM, Prazmowski. Motile, peritrichous ; includes some of the butyric bacteria. Genera with monotrichous and lophotrichous ciliation are unknown as yet. Sub-family 3. Plectridieae. Rods drum-stick-shaped during sporulation. Genus PLECTRIDIUM, A.Fischer. Motile, peritrichous; some butyric bacteria, the parasite of tetanus and a methane ferment. FISCHER D 34 TAXONOMY Family 3. SPIRILLACEAE. Vegetative cell cylindrical but spirally twisted. Fission always transverse. Genus VIBRIO, Miiller and Loffler. Very slightly curved rods, 'comma' shaped; motile, monotrichous. Vibr.'o cholerae asiaticae and numerous other vibrios of fresh and salt water. Genus SPIRILLUM, Ehrenberg. Cylindrical cells twisted in an open spiral ; motile, lophotrichous. Spirillum undufa, Sp. ntbruin. Genus SPIROCHAETE, Ehrenberg. Cells long and attenuated, spirally twisted with numerous turns ; cilia unknown ; the cell membrane is perhaps yielding. Spirochaete Obermaieri (remittent fever). Order 2. TRICHOBACTERINAE. Vegetative phase an unbranched or branched filament or chain of cells, the indi- vidual members of which break off as swarm-spores (gonidia). Family i. TRICHOBACTERIACEAE. (a) Filaments non-motile, rigid, enclosed in a sheath. Genus CRENOTHRIX, Cohn. Filaments unbranched and devoid of sulphur granules. Genus THIOTHRIX, Winogradsky. The same, but containing sulphur granules. Genus CLADOTHRIX, Cohn. Filaments branched, false dichotomy (includes Sphaerotilus). (b) Filaments motile, with oscillating and gliding movements, and devoid of a sheath. Genus BEGGIATOA, Trevisan. Containing sulphur. For the genus Streptothrix see next chapter. Further particulars as regards the discrimination of genera and species will be found in the works quoted in Appendix (No. 3). CHAPTER IV TAXONOMY (continued}. The Systematic Position of Bacteria; Other low Organisms with Pathogenic Properties. A QUESTION often asked is, whether bacteria are animals or plants'? Now the terms ' animal ' and * plant ' are collective terms invented by laymen to describe familiar living things, insects and elephants, mosses and oak trees, and they date from a time when such minute beings as bacteria were quite unknown. It is therefore as superfluous as it is futile to attempt, as many have done, to detect the distinguishing characters of the ' animal ' and ' vegetable ' kingdoms among organisms for which these terms were never intended. For this reason Haeckel and others have proposed to establish a third domain, that of the Protista, which shall include all those forms in which differentiation has not been pronounced on the lines of either animal or plant development. The new group would take up Radiolarians, Flagellata, and Infusoria from the animal side, and the Cyano- phyccae as well as some low forms of Algae and Fungi from the plants. The border line between protista on the one hand and plants and animals on the other, is, it must be confessed, artificial. To these protista, which embrace approximately all those forms of life we commonly call micro- organisms or microbes, the bacteria belong. Another question almost as common as the first is, whether bacteria are fungi, as the synonym Schizomycetes or fission-fungi would seem to imply. As far as the processes of life are concerned the bacteria and fungi agree in every detail, for, with very few exceptions*, the members of both groups are unable to derive their nourishment from inorganic compounds. That is to say both bacteria and fungi arc mctatrop/iic, are restricted in their food to substances fabricated by the higher organisms. Some of them arc even * The saltpetre bacteria and others. D 2 36 TAXONOMY paratrophic, that is, are able to exist only upon living animals or plants. But in spite of the many points of physiological similarity between fungi and bacteria there are very wide morphological differences. In every fungus, be it a mushroom, a morel, a mildew (Fig. 15, c}, a smut-fungus, or an animal parasite, like Herpes, we can always recognize two distinct parts, the mycelium or vegetative section, and, arising from this, the spore-bearing or reproductive section. The mycelium is in all fungi an irregularly felted mass of branched filaments, the hyphae, which in many cases (e.g. Penicillium, Fig. 15, c] consist of cylindrical cells set end to end, each cell having much the appearance of a rod-shaped bacterium. The fructification is of varying degrees of com- plexity, from isolated cells or chains of cells (Fig. 15, c) among the lower fungi FIG. 15. A, Oscillaria tennis (one of the Cyanophyceae\ fragment of filament ; c/t, hollow cylindrical chromato- phore ; c, so-called central body, finely vacuolated protoplasm with deeply-staining granules (black). B, Polytoma uve//a, flagellate with two anterior cilia ; v. contractile vacuole ; k, nucleus ; A, membrane, cell contents filled with assimilation products iparamylum). c, Penicillium glaitcum (true fungus, mycomycete), mycelium has arisen from the conidium a ; on aerial fiyphae brush-like fruits with chains of conidia. Magn. a 2250, 6 about 600, c (from Brefeld) 120. up to the highly complex sporophores of the toadstools and mushrooms. Upon a suitable soil the mycelium is able to vegetate for long periods, continually producing new reproductive cells or fruits. Among the bacteria such a differentiation is nowhere to be found. Their vegetative phase is either a single cell or a cell filament on which special reproductive organs are not developed but which, like Cladotkrix, breaks up entirely into gonidia. When the reproductive organs, the spores, appear, the vegetative cell as such ceases to exist. As in the myxomycetes and many other protista, it is entirely used up in the formation of the reproductive cell. The bacteria may therefore be termed holocarpons, as contrasted with the fungi which are eucarpoust that is to say, able to produce several successive fructifications from the same vegetative thallus. Failing to find the kindred of the bacteria among the fungi we must seek them among those low organisms which we have named collectively CYANOPHYCEAE 37 the protista. Of these, two groups in particular deserve careful attention, the blue-green algae or Cyanophyccac, and the Flagcllata. The Cyanophyccac present in their outward configuration many points of resemblance to the bacteria. \Yc find spherical cells (Chroococcus], rods (Aphanothccc], long cell chains or filaments (pscillaria), and spirally twisted unbranchcd forms (Sfirnlina). As among the bacteria, too, colonies or 'growth-forms' occur; some like sarcina (Glococapsa\ others in flat plates (Mcrismopocdia\ while sheath-bearing bacterial species like Cladothrix find their parallels among the Scytonenicae (Tolypothrix) with their dichotomous false branching. But it must not be forgotten that these striking similarities to the bacteria are not confined to the Cyanophyceae ; the chlorophyll- bearing algae offer a like series of parallels. The powdery green coating seen on the north side of tree-trunks, &c., consists of minute, free-living spherical cells (Pleurococcus)) pools and ditches are often coloured green by myriads of rod-shaped species (Stichococc2is\ the curved vibrios have their analogy in the gracefully- formed Raphidia, and examples of sheathed filaments and capsulation are not wanting. This is not surprising, for free-living cells must have the form either of spheres or cylinders, and the simplest cell unions must be filaments, flat plates, or packets. The similarity of external configuration is only a superficial similarity and in no way justifies a systematic approximation of the two groups. The cells of Cyanophyceae, whether free-living (Chroococcus, ApJiano- tJicce] or united to form filaments, multiply like all other cells by division, just as do the bacteria. Just as these, too, do the isolated Cyanophyceae 'split off' from their sister cells. And for this reason, supported by the not less superficial one of resemblance in shape, the bluish-green algae (• Schizophyceae ') have been placed alongside the ' fission-fungi' (' Schizo- mycetes') to form a separate class of plants, the ' Schizophyta/ But the ' fission' of these organisms is not peculiar to them, for it must always occur when isolated unicellular organisms divide; and the opinion that the bacteria are a series of colourless organisms parallel to the schizophyceae is not well founded. The differences between the two groups are as numerous as the similari- ties. The Cyanophyceae, if we except the gliding movements of the Oscillariac, are non-motile, whereas a large proportion of the bacteria are actively motile and possess special locomotive organs, the cilia. These arc moreover present not only at the time of reproduction, but persist tlirougJiout the life of the cell. The sporulation of the two groups is also different. The Cyanophyceae form not endospores but arthrosporcs, which arise by the differentiation and enlargement of a whole cell. In the finer structure of the cell again there is but one detail which the Cyanophyccac and bacteria have in common, the absence of a nucleus. In other respects the cells of the Cyanophyceae are much more highly differentiated than those of bacteria. 38 TAXONOMY The bluish-green colouring matter is borne by a special organ, the chromato- phore (Fig. 15, tf: cJi), generally having the shape of a hollow cylinder or sphere, forming the outermost layer of the cell-body and enclosing the remaining cell contents (Fig. 15, a, c\ The central mass stains deeply, and thus has somewhat the appearance of a nucleus. We are, however, no more justified in describing it as a nucleus than in calling the strongly-stained granules in the protoplasm ' nuclear chromatin.' As in the case of the ' chromatin grains ' of bacteria, the nature of these substances is unknown . In no bacteria, not even in the chromogenic species, is such a differentiation of the cell contents to be observed. If we now turn to the Flagellata we find among them also many points of similarity to the bacteria. In a species like Polytoma nvella (Fig. 15, b\ for instance, often found in stagnant waters, we have an egg-shaped cell enclosed in a definite membrane (//), and provided with a pair of permanent polar cilia or flagella. Other forms, such as Manas, are ' monotrichous,' others again, like Tetramitus, have a brush or tuft of cilia at the anterior end of the cell. The process of 'encystment,' too, closely resembles the formation of endospores, the cell-contents contracting and secreting a new membrane, the cyst or spore thus formed being set free by the disintegration of the membrane of the mother cell just as are the endospores of bacteria. But notwithstanding these points of likeness the Flagellata differ from the bac- teria in one fundamental feature : they possess a definite nucleus (Fig. 15, b : k) like that of the cells of Jiigher organisms. It is therefore evident that we have as little right to assume a close genetic relationship between bacteria and Flagellata as between bacteria and Cyanophyceae. The view most in harmony with observed facts is that which looks upon the Bacteria as a distinct group of protista, simpler in structure than the rest, and showing affinities to both Cyanophyceae and Flagellata. We might regard the bac- teria as the more direct descendants of the ancestral stock from which these arose, the differentiation of a chromatophore leading to the Cyanophyceae and the development of a nucleus as well as the general attainment of motile power giving rise to the flagellate phylum. Among the arche-bacterial types there may well have been both motile and non-motile forms which would have been respectively the starting-points of the two branches, whilst capsulation, the production of cell-chains or filaments, and other colonial or growth forms were primitive acquirements which have reappeared again among the Cyanophyceae and Flagellata and undergone greater specialization. The protista (micro-organisms), the simplest living things, among which we have tried to indicate the place of the bacteria, are not only manifold in form, but have very different modes of life and exercise a great variety of different functions in the economy of nature. As a general rule, these physio- logical features are not so conspicuous among the other groups as they are SACCHAROMYCES : AMOEJLIL 39 among the bacteria, but they become so when vigorous growth and suitable conditions of nourishment allow them to multiply rapidly. Such instances as the yeasts and the mould-fungi, with their energetic chemical effects, suffice to remind us that the bacteria are not alone in their performances. Some of these low organisms (22) are, like the bacteria, capable of pro- ducing pathological effects, but it is very rarely that such cases present the characters of a genuine infectious disease. Only within the last few years has it become known that the yeast-fungi (Saccharomycetes^ see Chap. XIV) are capable of developing pathological properties. Injection into animals of pure cultures of different species of brewery and distillery yeasts gives rise to processes having all the characters of true parasitic diseases, ending in many cases with the death of the animal. The germs spread to distant parts of the body and are found in all the organs as well as in the blood, but the lesions produced have not been recognized as those of any known disease in human beings. Still, an infection by yeast cells is just as con- ceivable as by any other organisms. It has recently been suggested that the various forms of cancer and similar neoplasms may be due to parasitic saccharomycetes, and some investigators have announced the discovery of such in stained sections. Unimpeachable results have however not been as yet attained, and it has been shown in some cases that the supposed para- sites were nothing but fragments of cells and nuclei of the tissues themselves. The parasitic nature of tumours is indeed denied by some pathologists. The thrush fungus (Saccharomyces alb leans of some authors) found on the buccal mucous membrane of children belongs possibly to the saccharomycetes. Its elongated cells have much the appearance of a yeast and they multiply by budding in the same way, giving rise to mycelium-like masses which cover the culture fluid with a thick pellicle. They set up, too, in suitable media (beer-wort) weak alcoholic fermentation. Whether these masses are true mycelia or not is uncertain, and it is therefore doubtful whether the fungus should be classed among the yeasts or with such hyphomycctes as Oidiimi and Monilia Candida. Some of the closely allied group of Flagellata occasionally occur as parasites or messmates in the animal body (e.g. MastigopJwra) which, as we have seen, have alliances to bacteria, but it has not yet been demonstrated that they are ever pathogenic. Trichomonas vaginalis is sometimes found, associated with other micro-organisms, in the mucous secretion of the vagina. A species called Trichomonas intestinalis has been found in the intestine in cases of diarrhoea and cholera, and a similar species occurs sometimes in the lung (sputum) in cases of bacterial infection. It is highly probable that all these forms are merely harmless water organisms which have found their way accidentally into the human body. Of more importance are some of the Amoebae, that group of naked protozoa which move about by the protrusion and retraction of portions of 4o TAXONOMY their protoplasmic substance to which the name pseudopodia has been given. The amoebae multiply rapidly by simple fission and, like many other unicellular organisms, undergo from time to time a process of encystment. The cysts are enclosed in a thick membrane which possesses great powers of resistance, and from them, under suitable conditions, the new amoebae emerge. Inasmuch as the amoebae are common in ponds, and ditches, and in damp earth, it is evident that they can obtain easy access to the human body. The best known pathological species, Amoeba coli, has been frequently described in cases of dysentery. But it is not present in all cases, and it seems to occur sometimes in the healthy intestine, so that its causal connexion with dysentery has yet to be proved. Pure cultures, free from bacteria, have not yet been obtained, nor has it been possible to cause infection by inoculation in animals. An amoeba-like organism of very doubtful legitimacy has been described associated with cow-pox (Cytoryctes -variolac], but it is certainly not the cause of the disease. The great prizes for the discovery of the vaccine microbe have yet to be won. Closely related to the amoebae is, no doubt, the Plasniodiuui malariac (Haemamoeba, Laverania], which occurs in the blood in cases of malarial fever. It is a very minute amoeboid organism which lives in the plasma, and (in greater numbers) inside the red blood corpuscles. At first colour- less, it becomes pigmented later on from the accumulation of granules of a black substance (Melanin] which arises from the decomposition of the haemoglobin. The plasmodia are seen to be most numerous during the periodical attacks of fever, one, three, or four days as the case may be, which characterize the disease. They then either become disintegrated or break up into a number of small spheres which have been called spores. Then the number of amoebae increases again till the next paroxysm. Whether these are true spores or not, is, like much else written about the malaria parasites, uncertain. They have not been seen to germinate, nor has it been possible to obtain pure cultures of the organisms. It seems, however, certain that they are really the cause of the disease, for its symptoms can be set up in a healthy body by the injection of blood containing the plasmodia. How the parasites enter the body, whether by the respiratory tract or the intestine, or, what is more probable, through minute wounds, such as insect-stings, is not yet known. Their habitat and mode of life outside the body are also unknown, but there can be no doubt that they are saprophytic in malarial districts *. In the blood of birds, reptiles and amphibia, there are sometimes found * .Researches now in progress on the west coast of Africa and elsewhere seem to show that certain species of mosquitoes are the bearers of the malarial parasites which are introduced into the human body by the insects' probosces. : LEPTUT1UUX. ACT1. \(>MYCLS 41 parasites similar to the plasmodia, with which they have been associated to form a new group, the Hacinosporidia. Their complete life-histories arc unknown, and clinical descriptions arc wanting. The best known species is Drcpanidiiiiu ranac, from the blood of the frog. Formerly described as a Cytozoon, it was at one time regarded with great interest, as it was thought to be not a parasite, but a tissue element of the frog. Its parasitic nature is now, however, generally recognized. There may be mentioned here the numerous kinds of parasitic micro-organisms, which are classed together as Sporozoa (Grcgarinae, Coccidia, Sarcosporidici}. They are all very imperfectly known, as it has not been possible to study them in pure cultures. Among the organisms that have been at different times reckoned as bacteria is a small group of fungi, some of them pathogenic, known as Streptothrix. They form felted mycelial masses of extremely delicate branched hyphae. In pure cultures they are in some cases sterile, but in others bear fructifications in the form of conidia, either single or in short chains, arising from the hyphae, resembling in this respect the lowest fungi (Haplomycetcs, HypJiomycctes), to which they undoubtedly belong. They have nothing in common with the bacteria. There seems to be reason to think that the genus Streptothrix (Oospora] will meet the same fate as the so-called Lcptomitns. Leptomitus was the name formerly given to all and every kind of filamentous fungal growth that made its appearance in neglected solutions in druggists' shops, in ink, &c., &c. We now know that these are not the complete forms of any specific fungus, but merely the sterile mycelia of various moulds growing slowly and abnormally in more or less unsuitable media. There can be little doubt that many growths now termed streptothrix will in like manner turn out to belong to well-known genera of hyphomycetes, which will complete their life-history when planted on suitable media. Even the best known species of Streptothrix, S. actinomyces (Actinomyces bovis], the ray-fungus, docs not seem to run through its entire cycle of growth in our artificial cultures. Its thallus consists of branched filaments made up of cylindrical cells just as in an ordinary mould-fungus, and it grows upon agar or blood-serum in the form of thick wrinkled mats with a ' pile '-like surface. This velvety coat is due to the aerial hyphae which rise vertically from the mycelium and break- up at their free ends into rounded cells like conidia. Whether these really are spores or not remains to be proved. The ray-fungus gives rise in cattle, and sometimes in man, to peculiar hard tumours, commonly in the tongue or jaw ('lumpy jaw '). These tumours suppurate sooner or later and often spread to other parts. Infection seems to be effected by the agency of sharp awns of rye and other cereals on which the fungus probably grows as a mould. They pierce the mucous membrane of the mouth, and carry the spores into the subcutaneous tissue. Attempts to 42 TAXONOMY convey the infection by the inoculation of pure cultures or pieces of diseased tissue have not been, successful. Microscopically the actinomycotic nodules are seen to consist of nests of densely matted mycelium with the surface layer of hyphae radiating outwards in all directions. The ends of the fila- ments terminate in club-shaped swellings, whose appearance is characteristic and unmistakable. They were formerly regarded as sporangia, but accord- ing to more recent researches are the result of a swelling-up of the cell-wall, or due to the deposit upon it of a gelatinous substance. They would there- fore seem to represent rather a degenerative phase of the fungus than organs of reproduction. A number of hyphomycetes are associated with various skin-diseases. Whether they are strictly parasitic, or whether they occur outside the living body in nature as simple mould-fungi, is uncertain. One of the commonest forms is TricJiophyton toiisitrans, the cause of a depilatory skin complaint known as Herpes. The mycelium is found in the epidermal scales and blisters, and grows well in artificial cultures, giving rise to chains of cylin- drical gonidia. In Favns, a skin complaint common to man and animals, another hyphomyccte is found, Ac/wriou ScJiocnlcinii. Some observers think that this form should be split up into a number of distinct species, others are content with one. Its morphology (in a botanical sense) has not yet been fully investigated, but it belongs undoubtedly to the group of the haplo- or hyphomycetes. The beautifully formed mould-fungi of the genus Aspergillus also occasionally invade the human body. Besides the stalked heads with their radiating chains of conidia, another kind of fructification, the pcritheda, is known. These perithecia show that Aspergillus belongs to the Pcrisporiaceae (Ascomycetes), and for this reason some investigators have looked upon all haplo- and hyphomycetes as imperfect ascomycetes. According to this view we must expect to find some day the ascospores and asci of forms like Achorion, TricJiophyton, and countless others. This is undoubtedly going too far. There undoubtedly exist simple moulds whose whole life- cycle is comprised in mycelium and conidia. Aspergillus itself lives, particularly in cultures, for years without forming ascus fruits. The conidia of the mould-fungi retain their power of germination in the dried state for very long periods. Three species at least of Aspergillus, A. fumigatus, A. niger, and A.flavns (all with coloured spores), are possessed of pathogenic properties. If a suspension of the spores be injected into a vein in the rabbit, mycelia grow in all the organs of the body, and the abscesses formed are rapidly fatal. Among birds, spontaneous infection is not uncommon, commencing generally in the respiratory tract, the ear or the eye. Human beings, too, are sometimes attacked, but the disease remains local, not spreading to other parts of the body. In spite of these facts it is uncertain whether the MUCORINE FUNGI 43 aspergillus is the original cause of the diseased condition, or whether it merely finds an entrance in places where the natural resistance of the tissues is already lowered by injuries such as the attacks of other micro-organisms. The spores are omnipresent, and accidental invasion is so easily possible that every case must be judged on its own merits. There remain to be mentioned as possible pathogenic organisms a few species of the genus Mncor, a mould-fungus of the order PJiycomycctcs. They arc much less highly organized than the Aspergilli. The mycelium consists of branched, ramified, non-septate hyphae, from which rise vertical branches (aerial hyphae) that bear the spore capsules (sporangia). When injected into the blood the spores of M. coryinbifer and of M. rJiizopodi- fonnis set up lesions similar to those of aspergillus spores ; but only one case of spontaneous infection has ever been observed in man. It is worthy of remark that those species of Mucor and Aspergillus which are capable of growing in the tissues of warm-blooded animals flourish only at high temperatures (37° C.), whilst those which are satisfied with less warmth grow in the tissues cither badly or not at all. CHAPTER V DISTRIBUTION AND ORIGIN Distribution of Bacteria ; Their modes of life ; Spontaneous Generation. IT would be impossible to give a more concise and, at the same time, accurate account of the distribution of bacteria in nature than is furnished by Goethe's lines in ' Faust ' : ' Der Luft, clem \Ya?ser wie der Erden Kntwinden tausend Keime sich 1m Trocknen, Feuchten, Warmen, Kalten ! ' While Mcphisto's haughty words : ' Halt' ich mir nicht die Flamme vorbehalten ; Ich hatte nichts Apart' s fiir mich' remind us that fire is the most powerful weapon that mankind possesses against bacteria. Unfortunately it is an engine of destruction that cannot always be employed. In discussing the question of distribution, it is necessary to discriminate first of all between actively growing bacteria and latent germs. Highly resistant bacterial spores, and the hardly less resistant dried vegetative cells of many species, occur everywhere upon the earth's surface and upon the animals, plants, and inanimate objects that cover it. They are, in short, ubiquitous. But living bacteria in active growth and multiplication only occur where certain conditions necessary to their welfare are fulfilled; above all, presence of water — the vital element of all organisms — a suitable tem- perature and a suitable supply of food. In nature such conditions are found in ponds or rivers contaminated by dead plants or animals, in dung, manure, and moist soil. In our dwellings it is chiefly in milk and other dairy produce, stale meat, and victuals of all kinds that bacteria are found. The majority of them, both in and out of doors, belong to harmless species, but there is no doubt that occasionally pathogenic forms occur. To find out the natural haunts of these dangerous neighbours, places where they are BACTERIA l\ EARTH. .-//A', AND ll'ATER 45 present not merely as isolated specimens but in large numbers, is one of the most important tasks bacteriology has before it. Methods for detecting bacteria in air, water and earth, have been greatly perfected during the last few years, but only the general principles can be treated of here : practical details will be found in works especially devoted to bacteriological technique (23). Atmospheric germs may be demonstrated in a rough-and-ready way merely by leaving exposed to the air nutritive culture media, such as agar or gelatine. The bacteria that fall upon the moist surface soon multiply, each one forming around itself a small colony visible to the naked eye. For an exact determination it is necessary to allow a measured quantity of air to pass slowly through a tube, the inner surface of which is coated with sterilized gelatine. Upon this the bacteria are precipitated, and the resultant colonies can then be counted and examined in detail. There are a number of other methods in which the air is drawn through sterilized glass beads, wool, sand, or sugar. The bacteria contained in it are thus filtered off, and the material containing them can be mixed with gelatine or other nutritive media, and the colonies allowed to develop in the usual way. The air in our dwellings usually contains more bacteria than that out of doors. Ten litres of air from a hospital ward, for instance, contained from thirty to a hundred germs ; the same amount of outside air only from one to five, half of these being the spores of fungi. Air in motion is always far more laden with bacteria than when at rest, and dusting or sweeping causes their number to increase enormously. That it must be so is self-evident, for dust of all kinds is rich in spores and dried but still living vegetative bacterial cells. They are whirled up into the air with the dust-particles which also bear adherent germs, and by reason of their lightness settle down through the atmosphere very slowly. Where there is no possibility for diseased secretions (e.g. sputum, diph- theritic membrane) to become dried and pulverized, the bacteria found in the atmosphere are of a harmless kind, or at most pus-cocci are present. The air expired from the lungs is absolutely free from micro-organisms, so that evidently the respiratory tract must act as a germ filter, the bacteria we inhale being retained within the body. But the majority of them are harmless, and moreover adhere to the mucous membrane of the nose, mouth, and pharynx, only a very small number ever getting as far as the lungs. Still, seeing that we inhale, on an average, five hundred litres of air every hour and, consequently, some fifty to two hundred and fifty germs, the danger of dust contaminated by disease products is very great. Water has always been looked upon as one of the chief agents in the spread of infectious diseases, and its bacteriological analysis is therefore a matter of the utmost importance. Most natural waters contain bacteria, and even the distilled water of our laboratories holds sufficient nutritive substance 46 DISTRIBUTION AND ORIGIN in solution to allow some to develop. This is, however, not surprising when we consider that thirty thousand bacteria only contain about T£oth of a'milligram of solid matter. Rain-water is contaminated with bacteria which it has taken up in its passage through the air ; one sample contained thirty- five germs per litre. The water of rivers, lakes and springs contains very varying numbers of micro-organisms, their abundance depending largely upon the amount of organic matter present. As soon as water is rendered impure by the influx of such substances as are carried into rivers by sewers and drainage canals, it ceases to be merely a liquid in which bacteria can retain their vitality for a long time unimpaired, and becomes a nutritive medium in which they proliferate with great rapidity. The water of the Spree for instance contained, above Berlin, on an average 6,140 bacteria per cubic centimetre, that taken below the city no less than 243,000. The bacteriological analysis of water is a very simple process. If the microscope shows that bacteria are not too numerous, a measured quantity of water (generally i c.c.) is mixed with nutrient gelatine and poured out on to a glass plate or shallow dish. The germs distributed through the gela- tine are allowed to develop into colonies which can then be counted in the usual way. The mere enumeration of the bacteria in water is of far less importance than the determination of their nature and properties. The majority of forms found in rivers and springs are of an entirely innocent character ; they are bacteria whose natural habitat is water, and they per- form a useful task in consuming the dissolved organic impurities. But when by any means pathogenic species obtain access to such waters these may easily become a source of infection, particularly when, as is the case with cholera and typhoid organisms, the bacteria belong to species which can live and multiply side by side with the ordinary saprophytic forms (see Chaps. XIII and XIV)*. When the water to be analysed contains a very large number of bacteria it must be diluted, before mixing, with an equal quantity of sterilized water. For the detection of isolated pathogenic forms in comparatively pure water, that is to say in water containing very few bacteria of any sort whatever, a method of ' nursing ' or ' enrichment ' is employed. A sterile nutritive solution (peptone-sugar) is added to the water, so that the few germs present may multiply before the gelatine is added. This method involves of course the drawback that the non-pathogenic species multiply also, and the danger that they may outnumber and overpower the less abundant bacteria we are looking for. Numerous aquatic bacteria are able to resist freezing for long periods, and ice sometimes contains enormous quantities of them (e. g. 2,000 per c.c.). [Even glacier ice is not free from them.] Of all the natural habitats of bacteria there is none in which they are * For marine bacteria, see Chap. VII. CLASSIFICATION ACCORDING TO NUTRITION 47 found constantly in such numbers as in the soil. They occur both as spores and in active vegetation (e.g. nitrifying bacteria). In the soil, as in water, the presence of organic matter is favourable to their increase, and they arc most numerous in cultivated earth. Garden mould seldom contains less than 100,000 germs per c.c., among them being almost always some pathogenic forms such as the bacilli of tetanus and malignant oedema. Putrefactive and pigment bacteria are also generally present as well as fermentative and nitrifying organisms. For the ordinary purposes of hygiene the culti- vation upon nutrient media of the bacteria of earth, water, and the atmo- sphere, and the detection and determination of pathogenic species, are sufficient ; the latter task being, however, by no means easy. Still more difficult and tedious is the systematic investigation of all the species present, necessitating as it does the employment of a number of different culture media varying in composition according to the nature of the species to be cultivated. It is not possible, for instance, to isolate the nitrogen-fixing bacteria of the soil upon a gelatine suitable for the tetanus parasite. In each case the character of the culture media must correspond to the require- ments of the organism to be cultivated. It has been customary to divide the bacteria, according to their mode of life, into two great physiological classes, the Saprophytes and the Parasites. Both groups are dependent upon other organisms for their sustenance ; both are unable either to build up their protoplasm from inorganic bodies, or to obtain, from the decomposition of such, the energy necessary for their vital processes. Those bacteria which can live and grow only within the tissues of living organisms are called parasites, those which are satisfied with the secretions or excretions of living, or the substance of dead, organisms, are called saprophytes. But this classification no longer corresponds to facts. Being a corollary of two axioms of general physiology, it could be held good only so long as their validity was unimpaired. The first of these axioms was, that of all organisms the green chlorophyll-bearing plants alone* arc able, with the aid of sunlight, to assimilate the carbon dioxide of the atmosphere, and from it to build up carbohydrates. All other living things, that is to say, plants devoid of chlorophyll, bacteria, and all forms of animal life, were supposed to be dependent, for their carbon, indirectly or directly, upon the carbon compounds already formed by green plants. The integrity of this principle has now been destroyed by the discovery of certain bacteria in the soil, which are able, even without sunlight, to appropriate the carbon dioxide of the atmosphere. The second postulate was that no organisms of any kind could fix and utilize the free nitrogen of the air. All organic nitrogen was thought to be derived from the nitrates of the soil ; green plants being * The red and the brown seaweeds also. 48 DISTRIBUTION AND ORIGIN again the only forms of life able to take it up in this state, other organisms receiving it from them at second hand in the shape of highly complex nitrogenous bodies. But this theory too has had to be abandoned since bacteria have been found, semi-parasitic in the tissues of plants, that can assimilate and store up free nitrogen (see Chap. X). The two groups of bacteria which necessitate a qualification of these two fundamental principles are, in a sense, complementary to one another ; on the one hand, the earth bacteria which get their nitrogen from soluble nitrates, and their carbon dioxide from the air ; on the other, the bacteria living on certain Legumi- nosae which derive their carbon dioxide from the tissues of the plant, and their nitrogen from the air. These facts force us to recognize the existence among bacteria of a special group of forms characterized by an extremely primitive metabolism, a physiological humility which shows them to occupy the very lowest rung of the ladder of life *. Such organisms as these cannot possibly be called saprophytes, and placed side by side with organisms which, like the putrefactive bacteria, have complicated physiological requirements. A better classification would be to divide the bacteria, according to their mode of life, into three biological groups, prototropJric, metatrophic t and paratropJiic (24). Prototrophic species are those which either require no organic compounds at all for their nutrition (nitrifying bacterium), or which, given the smallest quantity of organic carbon, can derive all their nitrogen from the atmosphere (bacteria of root-nodules). With them may be classed those forms that are able, with a minimal supply of organic matter, to break up specific inorganic bodies and derive energy from the process (sulphur and iron bacteria). In no case are the chemical changes involved clearly understood, and there apparently exist forms exhibiting gradations between the purely proto- trophic and the metatrophic habit. The metatrophic bacteria, under which heading most known species must be placed, cannot live unless they have organic substances at their disposal, both nitrogenous and carbonaceous. They flourish in every place where these are accessible, in impure water, on foodstuffs of all kinds, and in all the cavities of the body which are in communication with the exterior and offer nourishment in the form of secretions or particles of food ; the mouth, fot instance, and nose, the alimentary canal, and the vagina. Many of the metatrophic bacteria bring about profound changes in the chemical composition of the media in which they grow: fermentation (zymogenic bacteria), or putrefaction (saprogenic bacteria). Others break up the sub- stratum in a less conspicuous manner, but develop particularly in media * It seems more than probable that farther investigation will result in the discovery of other low organisms (protozoa) with similar functions. CLASSIFICATION ACCORDING TO NUTRITION 49 which have already been decomposed by saprogenic species, and contain a mixture of organic compounds which serve as nutriment. Such bacteria may be termed saprophilc. In the case of some metatrophic species the chemical change effected by their growth varies according to the nature of the medium they grow in (polytrophism), while the action of others is restricted to some one specific process (monotrophism, see Chap. III). The ability to grow in the living tissues is possessed in various degrees by different species, they are faculta- tive parasites (cholera, anthrax, and perhaps typhoid bacilli), but many are totally unable to exist under such conditions (obligatory sapropJiytes, obligatory metatrophic bacteria]. Finally, the paratrophic group includes all those bacteria that can exist only within the living tissues of other organisms. Paratrophic bacteria occur either not at all outside the animal body (gonococci), or else only as dust-borne spores or resting cells (tubercle, diphtheria). Their culture is possible only under conditions closely resembling those of the living tissues (on serum at blood-heat, for instance), and where there is freedom from competition with rapidly growing metatrophic species. Some paratrophic bacteria seem to flourish well even outside the animal body, if their growth on culture media is any criterion, but the conditions here are so very special that no conclusions can be drawn therefrom as to their behaviour in nature. Using then the modes of nutrition as a basis for classification, we distinguish the following groups:— I. PROTOTROPHIC BACTERIA. Nitrifying bacteria, bacteria of root-nodules, sulphur and iron bacteria ; occur only in the open in nature, never parasitic, always monotrophic. II. METATROPHIC BACTERIA. Zymogenic, saprogenic and saprophile bacteria ; occur in the open and upon the external and internal surfaces of the body, sometimes parasitic (facultative parasites), monotrophic or polytrophic. III. PARATROPHIC BACTERIA. Occur only in the tissues and vessels of living organisms, true (obligatory) parasites. It is worthy of remark that not only the bacteria but all other organisms can be arranged in these three biological divisions. All green plants from the simplest unicellular alga up to the largest forest trees are prototrophic ; all fungi and animals are metatrophic, except parasitic forms, which are paratrophic. Seeing that the spores of metatrophic bacteria are ubiquitous, it is not surprising that all liquids offering suitable conditions of nourishment should, if left uncovered and exposed to the air, soon become turbid from the FISCHEK E 50 DISTRIBUTION AND ORIGIN development of bacteria. But although it seems to us nowadays self- evident and unavoidable that such turbidity must owe its origin to the development of the ubiquitous and invisible germs — omne vivum ex ovo — this occurrence was, until not so very many years ago, a profound mystery. It appeared absolutely certain that these living ' animalcula ' must have arisen spontaneously from the non-living constituents of the liquid. ' Spon- taneous Generation ' (generatio acquivocci) seemed, on apparently good evidence, an indisputable fact. The question as to the origin of life — an older problem even than that of evolution — is the natural outcome of the Kant-Laplace theory of the origin of the earth, according to which our planet was at first a molten mass that had to cool down before living things could exist upon its surface. Whence came these first forms of life ? Were they precipitated from other planets through space upon our globe, or did they arise de novo from the non-living substances that then alone existed ? The first hypothesis has little to support it, and would only throw the question one step further back. It seems much more probable that the first organisms did really arise from inorganic matter by ' spontaneous generation,' and that, as the evolutionary theory assumes, all other living things have been gradually evolved from them in continued series. The doctrine of evolution is, indeed, incomplete without the assumption of such a commencement. Its possibility once granted, there is no a priori reason to deny that it still takes place, and that side by side with the ceaseless evolution of new species there may actually arise new organisms by spontaneous generation. Since we can imagine only the very simplest forms of life to be produced in this manner, it is natural that evidence for their occurrence should have been sought among bacteria and other fermentative organisms. Experiment showed that in some cases boiling for many hours did not prevent the development of bacteria in nutrient solutions, and since it was contrary to all experience that any living things could have survived such temperatures spontaneous generation seemed to be beyond dispute. When, as frequently happened, such organic infusions remained sterile after boiling, the advo- cates of the theory explained matters by saying that conditions had been changed, and the fluid was ' no longer suitable ' for the genesis of life. They also showed that passing air through it apparently removed these unsuitable conditions, bacteria appearing again. If, however, the air had been raised to a high temperature first, or passed through sulphuric acid, or even through cotton-wool, the sterility of the liquids sometimes remained unimpaired, but not always. Contradictory results abounded and difficulties arose both for partisans and opponents of the theory. It was long before the controversy could be regarded as satisfactorily settled, but settled it was at last, and we know now that at the present day a spontaneous generation of organisms does not take place in this way. We know that SPONTANEOUS GENERATION 51 in all those cases where bacteria appeared in liquids after protracted boiling they arose from highly resistant spores, whose vitality the heat had been unable to diminish, and we know further that those experimenters were right who sought to render air sterile by passing it over sulphuric acid or through heated tubes, and that it was for this reason that the infusions sometimes remained clear. Infusions of organic substances, if they be only boiled long enough, will remain sterile for years, even in vessels closed only by a plug of cotton wadding. A spontaneous generation of new life never takes place ; for bacteria, like all other organisms, can only originate from pre-existing germs. The fantastic theories that spring up from time to time regarding the derivation of bacteria from the disintegrated protoplasm of the higher creatures are apocryphal and, like the stories of organisms that consist only of carbon, oxygen, and hydrogen, must be received with the scepticism they deserve (26). Bacteriological research has thrown light from another side also upon the irrepressible theory of spontaneous generation. Before the discovery of prototrophic micro-organisms it was inconceivable how the first forms of life to appear upon the earth could have obtained their nourishment, for all organisms, including the infusoria and other protozoa, seemed to be metatrophic. Now, however, that we are acquainted with the meta- bolism of the prototrophic bacteria, particularly the nitrifying bacteria, we can form some conception of the mode of nutrition of the earliest terrestrial organisms E 2 CHAPTER VI PHYSIOLOGY OF NUTRITION AND GENERAL PRINCIPLES OF CULTURE Chemical Composition of Bacteria (27). THE substance of bacteria, like that of all other living organisms, consists for the main part of water, which constitutes about 85 per cent, of their weight (Jiuman body 65-70 per cent., green plants 60-80 per cent., algae about 90 percent.). The high proportion of water (shown also in the abun- dance of sap-vacuoles) is due to the fact that they are aquatic, not terrestrial organisms, inhabitants of liquid media. Analysis of the substance of pure cultures freed as far as possible from the culture medium has given the following results :— (NENCKI) (KAPPES) Putrefactive bacteria (a Bacillns mixture of several species). prodigiosus. Water 83-42 . . 85-45 Protein substances .... '3-96 . . i°-33 Fat i -oo .70 Ash .78 . . 1-75 Residue (not analysed) ... -84 .. 1-57 These two analyses give, of course, only a general idea of the composition of the bacterial cell. Other cases would probably show greater differences, for bacteria are, like all other organisms, dependent in their composition on the nature of the food-stuffs available. Like these also they are no doubt capable of selecting their food to a certain degree. The quantity of the ash will be larger where the surrounding medium is rich in salts, and there will be a higher percentage of proteids in bacteria from a nutritious peptone culture than in those nourished on glycerine and ammonium chloride. Still, on the whole, their composition is evidently not widely different from that of other organisms, as the above analyses show. CHEMICAL COMPOSITION OF THE CELL 53 Ncncki (27) isolated the protcid constituents of the bacterial cell by pre- cipitation with boiling hydrochloric acid, removal of fats with ether and alcohol, solution in caustic potash and final precipitation with chloride of sodium. The substance thus obtained (mycoprotein\ agrees pretty closely in composition with one prepared by Schlossbcrger from yeast, and contains 52-39 per cent, carbon, 7-55 per cent, hydrogen, 14-75 Pcr ccnt- nitrogen, and about 25 per cent, oxygen. Even if we assume that this body (free from sulphur and phosphorus) is an unaltered constituent of the bacterial cell and not a decomposition product from more complex proteins, it is not necessarily the essential constituent of the protoplasm. If it were we should have to place the bacteria chemically as well as morphologically below all other organisms, where, as far as we know, the phenomena of life are inseparably bound up with the presence of highly complex bodies like nuclein and nucleo-albumen rich in phosphorus. Since, how- ever, such compounds do undoubtedly occur in some bacteria, further investigations are necessary before we can form an opinion as to the nature and importance of mycoprotein. Closely related to the proteid bodies (using the word in its widest sense) are those poisonous substances known as toxalbumines. The constitution of their molecule is, however, as yet quite unknown*. Carbohydrates are probably present in all bacteria, but they certainly do not play such an important part as in the cells of higher plants, for, as already mentioned, the cell-membrane of bacteria consists not of cellulose but of a proteid substance. Apart from the not very clear indications afforded by the 'granulose reaction' in some few species (p. 13) there are no instances of differentiated carbohydrate cell-contents. The gelatinous substance produced by Leuconostoc (see p. 10) and by some of the mucilaginous species that occur in wine and beer is probably a carbohydrate (dextrane, [CGH10O5]n) similar to cellulose and the slimy secretions of some gelatinous algae. As special temporary cell constituents must be regarded also those products of fermentation which, although formed in the cell, are not stored up but excreted as metabolic products (see Chaps. XI-XIV). The percentage of ash given in the various analyses of bacteria cannot be taken as indicating in any degree the value of salts in the economy of the cell, because the nutritive media were not prepared with this end in view. Food Stuffs of Bacteria (28). Mineral substances are as necessary for the Bacteria as for all other organisms, albeit in a much smaller quantity. One milligram of living bacteria (i. c. about thirty milliards of individuals) with I per cent, of ash * For the relations of the toxalbumines to infectious diseases, see Chap. XVII. 54 PHYSIOLOGY OF NUTRITION would contain only y^ milligram of mineral salts, so that it is evident that very minute quantities of such salts are sufficient for nutritive media, say from 01 to 0-2 per cent. Of the elements may be mentioned as indispensable : sulphur, phos- phorus, calcium, magnesium, potassium, and sodium, with traces of chlorine and iron. Whether other alkalies such as rubidium or caesium can be substituted for potassium and sodium, or other alkaline earths, such as barium or strontium, for calcium, is doubtful. Recent experiments on the metabolism of mould-fungi render it improbable. Full details as to the most suitable proportions of mineral salts in nutri- tive media will be found in practical treatises. In the following brief account of the nutrition of bacteria, the words ' necessary salts ' will be understood to mean o-i per cent. K2HPO4, 0-02 per cent. Mg SO4, and o-oi per cent. CaCl2. Sodium and iron are also necessary, but are always present in traces in the other chemicals used unless these have been specially purified ; they are likewise present in tap-water. For pathogenic forms the addition of from 01 per cent, to 0-7 per cent. NaCl is advisable ; but if the media have been prepared with infusion or extract of meat it is not necessary. These few mineral substances will be found sufficient probably for all bacteria, be they prototrophic, metatrophic, or paratrophic. The require- ments in carbon and nitrogen compounds, on the other hand, are widely different for each group. The prototrophic nitrifying bacteria grow well in a fluid of the following composition :— Water, H2O loo- Potassium Nitrite, KNO2 .... -05 Potassium Hydrogen Phosphate, K.2HPO! . -02 Magnesium Sulphate, MgSO4 .... -03 Sodium Carbonate, Na2CO3 .... -05 Sodium Chloride, NaCl -05 The nitrous acid supplies the nitrogen, and the carbon is derived, not from the Na2CO3, but from the carbonic acid of the atmosphere. Other proto- trophic soil bacteria are able to fix atmospheric nitrogen, but require some organic compound as a source of carbon ; sugar for instance. The growth of various metatrophic bacteria upon solutions of different carbon and nitrogen compounds is illustrated by the following table. All the solutions contained the same amounts of mineral substances, and, with the exception of three cases, had an alkaline reaction ; free acid, as the figures show, generally retarding development. The cultures were kept at the most suitable temperature in each case and were watched for fourteen days, so that delayed growth might not be overlooked. The symbols employed have the following meanings : — FOOD STUFFS 55 -\--\ — (- = very luxuriant growth, liquid turbid with bacteria or a pellicle upon the surface (cholera, />'. sttbtilis'), or deposit of bacteria at bottom of vessel (anthrax). H — \- = growth moderate, faint but distinct turbidity or a delicate pellicle. + = feeble growth, liquid opalescent or turbidity visible only on shaking. + ? = scarcely any growth. O = no growth. Source of Nitrogen. Source of Carbon. Reac- tion. B. An- thracis. B. typhi. B. coll. vibr. cholerae. B. subtilis. B. pyo- cyaneus. 1 ]% Peptone i % Grape sugar alk. +++ +++ ++ + +++ +++ +++ 2 „ Peptone ,, Peptone alk. ++ ++ ++ ++ + +++ 3 „ Asparagine „ Grape sugar alk. O + +++ +++ +++ +++ 4 ,, Asparagine ,, Grape sugar acid O + ? +++ 0 + + +++ 5 ,, Asparagine „ Asparagine alk. O o ++ +4- + + 6 ,, Asparagine „ Asparagine acid 0 O + o + -f 7 ,, Ammonium tartrate ,, Glycerine alk. O o ++ + +++ +++ 8 ,, Ammonium tartrate ,, Ammonium tartrate alk. O 0 + ? 0 O + ? 9 ,, Ammonium chloride „ Glycerine alk. O o +++ ++ ++ ++ 10 „ Ammonium chloride ,, Glycerine acid O o +++ o ++ + 11 ,, Potassium nitrate „ Grape sugar alk. 0 0 + +? ++ +++ 12 ,, Potassium nitrate ,, Glycerine alk. 0 o O o o +++ 13 none ,, Sugar alk. 0 o o o o 0 ? U „ Potassium nitrate none alk. O 0 0 0 0 o The most conspicuous result that the table shows is the great difference between B, anthracis and B. pyocyaneus. The former prospers only where peptone is available as a source of nitrogen ( i and 2), the latter grows just as well and exhibits its magnificent fluorescence upon nitre (12). In other words B. anthracis is a 'peptone bacterium} B. pyocyanens a ' nitrate bac- terium ' approaching the members of the prototrophic group in its physio- logy. The latter, however, needs a special source of carbon, not being able to assimilate the carbon dioxide of the air (14). There is another numerous group of metatrophic forms which, given carbon in a suitable form, can utilise the nitrogen of ammonia compounds, and thrive as well as they do upon peptone. B. coli is one of these ' ammonia bacteria, and also V. cholerae and B. subtilis. Others, B. typhi for instance, are ' amido- bacteria ' ; they cannot assimilate the nitrogen of ammonia, but grow fairly well upon amide bodies, such as asparagine and leucine. One fact must not be lost sight of if the bacteria are classified in this way, according to 56 PHYSIOLOGY OF NUTRITION their nitrogen requirements: the power of utilising the nitrogen in different compounds is largely dependent upon the form in which the carbon is supplied. Thus, the ammonia bacteria can utilize the nitrogen of saltpetre if sugar is present, but they are unable to do so if the carbon is presented to them in the form of glycerine. ATo bacteria can dispense with nitrogen entirely, and the feeble growth of B. pyocyancus in sugar solution alone (13) was probably due to the presence of impurities, or to the absorption of ammonia from the air of the laboratory. The first attempts to characterise bacteria according to their behaviour towards different nitrogen compounds were made by Nageli, who also examined yeasts and mould fungi in the same way. The subject has been still more recently studied by Beyerinck (~8). In medical bacteriology, fluid culture- media are now very rarely employed, having been driven out by the stereotyped recipes for peptonised gelatine and agar. That this has been disadvantageous there can be no doubt. The case of B. typJii and B. coli, the one an amido-bacterium, the other an ammonia bacterium, shows the value of fluid media of varying composition for the differentiation of species. Not less important are the conclusions which may be drawn from such investigations as to the occurrence of pathogenic bacteria in nature. This subject will be more fully discussed in later chapters. The form in which nitrogen is offered affects greatly the influence which the reaction of the culture medium has upon the growth of the bacteria. The more assimilable the nitrogen compounds, the less sensitive are the bacteria to the deleterious effect of free acid. B. pyocyaneus, as the table shows, is, when it gets its nitrogen in the form of asparagine (5-6), unaffected by the acidity of the medium which palpably retards growth when ammonium chloride is the only source of nitrogen. The extremely sensitive V. cholcrae is totally overpowered by free acids (4 and 6), while B. snbtilis, which grows well on weakly acid hay-infusion, is less marked in its behaviour, and B. coli seems to be quite uninfluenced by the reaction. The formation of pigment too, in B. pyocyaneus, as in other chromogenic bacteria, is largely dependent upon the reaction of the medium and the nature of the nitrogenous bodies it contains. The organic compounds of nitrogen, although they all of them contain carbon also, are not in themselves sufficient for vigorous growth, as a comparison of Nos. i, 2. 3 and 5 of the table shows. The carbon of ammonium tartrate (8), for instance, is absolutely useless to B. subtilis^ and even B. coli and B. pyocyaneus are half starved when they have no other carbon-containing compound to draw upon. The value of a special source of carbon, so clearly shown in the table, is twofold : the excess of organic carbon is used in the building up of proteids ; and, more important still, the breaking down and oxidation of FOOD STUFF* 57 the carbon compound provides the necessary energy for the full utilisation of inferior nitrogenous .substances (u and 12). The organic carbon compounds are of very different degrees of import- ance as food-stuffs, their value depending mainly but not exclusively upon their heat of combustion. Grape sugar and sugars in general are the best, then glycerine and other polyacid alcohols, such as mannite and dulcite, and finally compounds like tartaric and succinic acid, benzoic acid, and the monacid alcohols and their derivatives such as fatty acids and amines*. Compounds, in which an oxygen atom is linked by t\vo valencies to the carbon atom, as in urea CO(NH2)2, or oxalic acid (COOH)2, are useless as sources of carbon, as are also the cyanogen compounds. It would appear, therefore, that the carbon is in its most utilisable condition when it is linked with hydrogen in the form CH2. being less valuable as CH, still less so as CHOH, and not at all in the CO or CN radicles. Exceptions to this rule, however, must not be forgotten. Speaking generally, the best data for the discrimination of a bacterial species from the standpoint of nutritional physiology are those afforded by its behaviour towards nitrogenous compounds, the demands made for carbon being less narrow and well defined. The paratrophic bacteria are, as our table shows, able to grow only in those solutions that contain peptone. They approach in this respect most closely to the metatrophic forms, but their requirements are often much more special. The gonococcus, for instance, grows only upon albuminous media, such as coagulated blood serum. Upon this too the diphtheria bacillus thrives best. The tubercle bacillus, which we are obliged for the present to regard as a strictly parasitic species, is able to grow upon inferior substrata, even on those of the ammonia bacteria (see Chap. XVI). For the culture of bacteria there are commonly used, besides bouillon and other fluids, the so-called solid media prepared with gelatine or agar. The commonest recipe is an infusion of meat (i Ib. meat to I litre water), with about 1-2 per cent, peptone and sugar, to which 10 per cent, gelatine or 1-2 per cent, agar is added, the whole being boiled and filtered hot. This gives a clear gelatinous mass, in which the nutritive substances are equal ly distributed throughout the non-nutrient gelatine or agar. The introduction of such culture media (29) has been an important factor in the rise of bacteriological science, for it is only by the use of easily melted and easily congealed substrata that we are able to isolate bacteria without difficulty, and obtain them in pure cultures. The nutritive fluids mentioned in the table on p. 55 may of course be mixed with gelatine or agar, and the advantages of several methods united. * For details, a re-examination of which is desirable, see Nageli ; appendix No. 28. 58 PHYSIOLOGY OF NUTRITION If it is desirable to exclude all organic compounds, colloid silica may be used as a matrix instead of gelatine. Great latitude is permissible in the composition of the peptone-sugar-meat solutions that are used. Almost every laboratory has its own special recipes, dictated either by whim or by the results of long experience. In many cases the media are unnecessarily overloaded with nutriment. Besides meat, hay, straw, plums, potatoes and many other substances may be used to make nutrient infusions. The wort of beer is often used, and, as an opaque solid culture-medium, potatoes are often employed. For practical details the technical handbooks must be consulted. Apart from characters such as pigmentation or the production of gas, the modes of growth of different bacteria on the same substratum are sufficiently varied to be of some use in the differentiation of species. Too much importance, however, must not be attached to these features. In fluid media, two chief types of growth are seen. In the one the liquid remains clear, in the other it becomes turbid. When the fluid remains clear we know that we have to do with non-motile bacteria with a tendency to filamentous growth. They collect on the walls and bottom of the tube in flocculent masses, which may be dislodged by shaking (Anthrax, Strepto- coccus). When such non-motile forms need much oxygen (B. tuberculosis for instance), they form a thick membrane, sometimes smooth, sometimes wrinkled, over the surface of the clear liquid. All isolated, non-filamentous forms, particularly the motile species (Cholera, Typhoid), cause the medium to become uniformly turbid. The turbidity varies from a dense milky opacity to a faint opalescence only visible on shaking. Strongly aerobic species form a pellicle on the surface of the fluid ( V. c/wlcrae, B. subtilis). By culture upon gelatine we are able to divide the bacteria into two groups, those which, by means of a peptonising enzyme, liquefy the gelatine and those which do not. The great majority of known forms belong to the former class ; B. typhi, B. coli, Streptococcus, Lactic bacilli, &c., are examples of the latter. Further differences of growth on gelatine are brought out by the methods of ' plate ' and ' stab ' culture. In plate culture the melted gelatine is inoculated with a minute quantity of bacteria, or of substances containing bacteria, and then poured out in a thin layer upon glass. There it coagulates, and the germs it contains multiply. The germs being fixed produce around themselves sharply-defined ' colonies, each colony having arisen from a single germ or from a few stuck together. The young colonies in particular show many characteristic features ; colour, form, and contour, consistence and lustre, and the structure as seen under a low- power lens are all useful points. A detailed account will be found in Lehmann and Neumann's atlas (30), and is needless here. The following description of the colonies of two well-known bacteria will show the nature of the differences involved better than an enumeration of all the known MODES OF CULTURE 59 varieties of growth. The two species selected arc the cholera and the anthrax bacteria. The anthrax colonies liquefy the gelatine slowly; they are whitish and round ; under a low power the edge of the colony appears filamentous, in ' tresses ' and loops, not smooth ; in older colonies an irregular skein or clump lies in the fluid, almost transparent, gelatine. The cholera colonies liquefy the gelatine rapidly ; they are yellowish white, and magnified show a granular texture and an undulating, not filamentous, edge ; the liquefied gelatine immediately round the disintegrating granular clumps is turbid. It must not be forgotten that many variations occur, and that closely similar forms, such as the cholera vibrio and the aquatic vibrios or typhoid bacilli and B. coli, are not distinguishable by their colonies alone. The gelatine stab-culture is made by thrusting vertically into a tube of coagulated gelatine a platinum wire charged with the substance of a pure culture. Species which need oxygen grow only in the upper parts of the gelatine, anaerobic forms only in the deeper parts. Growth in filaments or chains shows itself by delicate threads growing out into the surrounding gelatine so that the path of the needle appears feathery or moss-like, e. g. Anthrax. Bacteria whose cells are isolated are confined in their growth to the path of the needle. Among species which liquefy the gelatine great importance is laid upon the shape of the liquefied tract. This may be equal all along the path of the needle, and have the appear- ance of a straight liquid tube in the middle of the solid gelatine, or, if liquefaction is more rapid towards the surface, a funnel-shaped well is produced. Here again considerable variations take place, and closely allied species are not distinguishable by these data alone. The surface of agar or gelatine that has been allowed to solidify in tubes in a slanting position is used for ' streak ' cultures. These are made simply by smearing the surface of the substratum with a platinum wire charged with bacteria. The differences in colour, contour, lustre, and con- sistence are similar to those of the colonies in plate cultures. Agar is not liquefied by any bacteria. The modes of nutrition of particular biological groups among bacteria will be considered in the subsequent chapters. CHAPTER VII RESPIRATION OF BACTERIA Aerobiosis and Anaerobiosis ; Light-producing Bacteria ; Marine Bacteria ; Sulphur and Iron Bacteria. THE old and now obsolete term ' vital air ' applied to oxygen meant that without it life was impossible. Of all the axioms of physiology, none has ever seemed to be so unassailable as that which asserted the necessity of oxygen to every living thing. It appeared to be a law absolutely without exception that all animals and all plants must ' breathel that is to say, must take up free oxygen in order to oxidise and break up the organic compounds of their food or of their tissues, and thus obtain the energy necessary for the processes of life. Deprivation of air seemed inevitably followed by cessation of life. Here again the study of yeasts and fermentative bacteria has overturned the old views and revolutionized our ideas regarding the chemistry of life. In 1861 Pasteur (31) discovered that many zymogenic bacteria could live and even set up active fermentation in the absence of oxygen ; and he gave them for this reason the name of anaerobes. His statements, romantic and improbable as they at first appeared, were soon corroborated, and the general recognition of their accuracy has resulted in the division of the bacteria into two groups, the aerobes and the anaerobes. In the aerobic bacteria the process of respiration is the same as in all ordinary organisms. They absorb oxygen, and with it break up non-nitrogenous bodies such as glycerine or sugar into carbonic acid and water. They are also able, like plants and animals, to assimilate nitrogenous substances such as peptones and amido compounds, although with less gain of energy and less easily than they can carbonaceous bodies. Many of the aerobic bacteria are totally unable to live without oxygen, and when deprived of it die as would a mouse in pure hydrogen. They are exclusive or obligatory aerobes. In rarefied air or in artificial mixtures of gases their vitality is proportional te the amount of oxygen present. Under an air-pump, for instance, they A ERG BIOS IS AND ANAEROBIOS1S 61 ceasi to grow long before a vacuum is reached (acetic bacteria, B. sub tills}. Contrasted with the obligatory aerobic bacteria, we have the obligatory anaerobic forms which thrive only in the absence of oxygen, small traces of this gas being sufficient to inhibit growth (B. tetani, some butyric bacteria, bacillus of malignant oedema). Between these extremes there is a great host of bacteria representing every gradation between the two modes of life. These are the facultative anaerobes, which, while growing best with a plentiful supply of oxygen, are nevertheless able to exist with a very small amount, and even with none at all, although in this case their vitality is often much impaired. Anaerobic bacteria, both obligatory and facultative, are found everywhere in nature where the air cannot penetrate, or where it is replaced by other gases —in the deeper layers of the soil, for instance, in the mud of rivers and standing waters, or the ooze of the sea bottom, and in manure. In all such places anaerobic bacteria are the principal and often the only forms of life, and by the fermentative and putrefactive processes they set up they effect the disintegration and removal of dead animals and plants.* As be- longing to the group of facultative anaerobic bacteria may be mentioned the lactic acid ferment, the bacteria of typhoid and cholera, many staphylococci and streptococci and most putrefactive bacteria. The power of anaerobic growth varies, even in the same species, according to the source whence the germs were derived and the mode of culture. Deprivation of oxygen affects the bacteria in many ways. Some of the pigment bacteria (B. vio- laceus, for instance) give rise to colourless cultures if oxygen be excluded ; others, like Spirillum rubrum, form pigment most plentifully under these conditions, though here again, as in so many other cases, exceptions occur. Many obligatory anaerobic bacteria (e. g. some of the butyric ferments) are motile, the energy necessary for movement being derived from the partial breaking-down of the molecules of the fermentable substances on which they grow. Access of oxygen stops the motion at once, just revers- ing the conditions found among the aerobic forms, where the cilia work the more vigorously the more oxygen is present, and become paralyzed at once if it be cut off. These movements, minute as they are, represent an expenditure of energy that is considerable when the size of the organisms is taken into account. Those aerobic bacteria which possess the power of movement are attracted by oxygen. If a drop of water containing them be placed upon a slide and covered with a thin glass slip, they crowd to the edges of the preparation and around air-bubbles in the water. This property of being attracted by oxygen has been utilised by Engelmann (32) for the detec- tion of minute traces of free oxygen. His ingenious method, which * The chemical aspects of the phenomena of putrefaction as well as the theoretical explanation of anaerobiosis are discussed in Chap. XIV. 62 RESPIRATION OF BACTERIA a B C .-v: employs aerobic bacteria as living reagents, has given us what has hitherto been unattainable, a micro-chemical test for the gas. It is a well-known fact that there is in green plants, going on side by side with the ordinary respiration of the cells, a second process of gaseous exchange accompany- ing the assimilation of carbon dioxide and also often incorrectly called ' respiration.' The carbon dioxide is split up in the green parts of the plant by the energy of the sunlight, the carbon being retained, and the oxygen thrown off into the atmosphere. Now it is not all rays that are equally powerful to cause this chemical change. The absorption spectrum of chlorophyll shows that the red rays between Frauenhofer's lines B and C are the most strongly absorbed by green plants, and after them that part of the spectrum just beyond F. It is just these regions that are most active in causing the evolution of oxygen, and Engelmann demonstrates this in the following way. By means of an arrangement of prisms in the substage of a microscope he pro- jects a spectrum upon green algal filaments or moss-leaves lying in the field of view, the water in which they are mounted containing aerobic bacteria. If, now, the illumination be strong, and all extraneous light be carefully excluded, it can be seen that the aerobic bacteria crowd round the filaments just where the active rays fall, the chief swarm lying between B and C, and an- other smaller one near F (Fig. 16). Around these parts of the alga only a few scattered organisms occur, show- ing that the evolution of oxygen is at a minimum. The application of this bacterial method for the detection of minute traces of free oxygen is an operation of great delicacy, and the results which it gives must be interpreted with great care because the movements of bacteria are very often rendered more active by other substances beside oxygen, particularly by food-stuffs, as will be explained in the section on chemotaxis. The attraction by oxygen is in fact only a special instance of chemotaxis. The great amount of energy derived from the combustion of food-stuffs, and from the oxidation of the tissues in higher organisms, is not all expended in the form of mechanical work, but applied in part to raising the tempera- ture of the tissues. In animals this is conspicuous in the case of ' warm- blooded ' creatures, and in plants we have instances in the germinating grains of cereals and in the flowers of the Aroideae, which are often sensibly warm to the touch. Fermenting and putrefying substances also (hay, manure, cotton- FlG. 1 6. Detection of oxygen by means of bacteria. The vertical lines are the Frauenhofer lines of a spectrum thrown on the field of the microscope. In the spectrum lies a filament of the alga Cladophora, and around it, at B, C, and F, the bacteria swarm (see text). Magn. 200. THERMOCENIC AND PHOSPHORESCENT BACTERIA 63 waste) frequently become heated, the temperature inside the mass being often raised to 60° or 70° C. This spontaneous heating, which may increase to actual ignition (spontaneous combustion), is due to the respiratory activity of aerobic bacteria (thcrmogcnic bacteria of Cohn), which set up fermentation and putrefaction. Cohn (33) found in damp cotton-waste a micrococcus which, when well supplied with air, raised the temperature of the decaying mass to 67° C., if care were taken to prevent radiation, carbonic acid and trimethylamine arising as respiratory products. Light is another form in which the surplus energy of respiration exhibits itself both in terrestrial and marine plants and animals. The existence of phosphorescent insects is a matter of common knowledge. The mysterious light often seen in old willow-trees is given off by the mycelia of certain parasitic fungi. Among marine organisms there are very many kinds which are luminiferous. The phosphorescence of the sea is mainly due to light- producing infusoria, hydrozoa, and ascidia, and in our latitudes above all to bacteria (34). The various kinds of phosphorescent bacteria have been collected together to form a ' biological ' genus Photobacterium. They include actively motile straight rods and vibrio- like forms, but very few have been completely described, and names like B. phosphorescent, B. luminosus, Vibrio albensis must not be regarded as designating ' good ' species. The faint glow seen upon decaying haddocks, mackerel, and other sea fishes, is pro- duced by these micro-organisms. Whether any phosphorescent bacteria occur in fresh water is doubtful : but all with which we are as yet well acquainted are from the sea. Being marine organisms, the phosphorescent bacteria need in their culture-media from two to three per cent. NaCl, besides the usual salts and peptone, and to obtain successful cultures of phosphorescent bacteria the media must contain these substances, besides some other source of carbon such as sugar, glycerine or asparagine. Phosphorescent bacteria would seem therefore to be ' peptone ' bacteria, saprophytic in the sea upon dead animals and plants, from which they are washed off in countless numbers by the waves. The phosphorescent bacteria of the North Sea and Baltic flourish best at about 18° C., but can grow quite well at much lower temperatures, even down to o° C., resembling in this respect the other inhabitants of northern waters. In the absence of oxygen they are indeed able to grow slowly, but are then not phosphorescent, the production of light being an exclusively aerobic pheno- menon. This is evident, too, when we watch the phosphorescence of the sea, for perfectly smooth water does not shine at all, while the brilliancy of the light is always greatest upon the crests of the waves, or where the water is churned up with air in the wake of a ship. That the respiratory exchange (oxidation) of the bacteria is the cause of the phenomenon is proved by the fact that the death of the micro-organisms or exclusion of 64 RESPIRATION OF BACTERIA oxygen quenches the light at once, whilst a more abundant supply of highly combustible food-stuffs like carbohydrates increases it. We know of no specific substance (luciferiri) to which the luminosity could be due, and the phenomenon is of quite different nature from that exhibited by phos- phorescent bodies like calcium sulphide, for in the bacteria the production of light is quite independent of previous insolation. Phosphorescent bacteria may easily be obtained by placing the flesh of fresh haddocks or herrings in a solution of two to three per cent, of NaCl and keeping at a low temperature (5°— 10° C.). In one or two days not merely the fish, but the whole of the liquid in which they lie, give off a pale-greenish light which becomes much more brilliant if a little sugar or glycerine (i. e. respirable material) be added. The bacteria may be readily isolated by the usual methods upon peptone-sugar-gelatine prepared with an infusion of fish and common salt. The pure cultures thus obtained are so strongly phosphorescent that by protracted exposure they may be photographed in their own light. The rays given off are only the more refrangible, from D to G. This is evident, too, from the bluish colour of the light. A few words may be said here regarding other marine bacteria (35). The investigations of the German Plankton Expedition show that the prevailing forms are actively motile rods and vibrios, cocci being less numerous. Their distribution is determined mainly by the proximity of land, for the algal vegetation which is richest near the shore attracts myriads of marine organisms that provide the bacteria with a rich variety of nutriment. The influence of the shore extends four or five miles out to sea. The degree of illumination seems without influence, and no regularity could be observed in the distribution, but the number of bacteria per c.c. varies immensely, however far from land the sample be taken, as the following figures show : Plymouth Docks (ebb) 13,320 per c.c. I mile from shore (flood) 3>96o „ 240 miles from shore (Gulf Stream) . . . 645 ,, 450 miles from shore (Sargasso Sea) . 20, 200, 206, 168 „ These numbers include the spores of mould-fungi. In 54 per cent, of all samples about 100 germs per c.c. were found ; at depths of from 800 to 1,100 metres below the surface only from eight to twelve bacteria were present. Specimens of ooze from the ocean bed at depths varying from 1,523 to 2,406 metres were sterile ; others again from 4,099 to 5,250 metres contained from one to four bacteria per c.c. This paucity is the more remarkable when we consider that the temperature was from 2° to 5° C., and the conditions altogether such that foraminifera and radiolaria occurred abun- dantly. Possibly the culture-medium (peptonized fish-broth gelatine) was SULPHUR BACTERIA 65 at fault, since it affords nourishment only to mctatrophic forms, not to prototrophic, and it is just these latter that we might expect to find on the ocean floor. It is not at all unlikely that there are species characterized by an extremely simple type of metabolism, which would throw new light upon the mysteries of marine life. Russel has discovered aerobic bacteria that reduce nitrates in samples of mud from the sea-bottom. In the aerobic bacteria which we have been considering, respiration consists essentially in the oxidation of complex organic substances, the energy thus set free being necessary for the processes of life. In the case of the anaerobes the energy developed, although smaller in amount, is likewise derived from the splitting-up of fermentable and putrescible organic substances. Both classes, aerobes and anaerobes, are metatrophic in this respect. There is, however, in certain prototrophic bacteria a process going on comparable to respiration, but consisting in the oxidation of inorganic compounds. The micro-organisms in question arc the nitre bacteria (see p. 105) and the remarkable sulphur bacteria, the classical examples of prototrophic re- spiration. The sulphur bacteria (36), Tliiobacteria (p. 13), whose cells are often crammed full of spherical refringent masses of pure sulphur, occur in nature in places where free sulphur- etted hydrogen is present. Such are sulphur springs where the SH., is principally of mineral origin, and the mud of standing waters and of the sea-bottom, where it is set free by the putrefaction of dead plants and animals *. The bacteria were formerly thought to produce the SH.,, and therefore to play an important part in the origination of sulphur springs, but the splendid researches of Winogradsky have shown that the micro-organisms need the gas as a food-stuff, and are unable to live without it. Thiobacteria can be found at any time of the year, but are most abundant in the early spring and late autumn, periods when the remains of the summer's vegetation in standing waters are plentiful, and when the putrefactive processes set up by other bacteria cause an evolution of SH2. The sulphur bacteria frequently form a snow-white furry coat on the rotting vegetation, with here and there pink or pale puce-coloured patches. FlG. 17. Sulphur Bacteria, a-c, Bezgiatoa. a full of sulphur (black rings) ; 6, partly S°° Caustic soda Ij5°° Cadaverin (bacterial toxine) ..... JjS00 Quinine hydrochlorate 500 Carbolic acid 500 Thymol 250 Salicylate of soda ....... 1 50 Alcohol 15 Common salt 15 The extraordinarily small quantities which are in some cases sufficient to COMPARATIVE EFFICACY OF DISINFECTANTS prevent putrefaction do not kill the bacteria, they only arrest growth and multiplication. II. LETHAL VALUE FOR SPOKELESS TUBERCLE BACILLI. % Inferior lethal coefficient ; the time is given in which tubercle bacilli (from cultures, not in sputum) are killed. From Yersin (54). Carbolic acid ,, ,,..... Absolute alcohol .... lodoform Ether Corrosive sublimate Thymol Salicylic acid To kill the bacteria in sputum, where they are embedded in mucus, a much longer time is necessary, e.g. 10 per cent, lysol for twelve hours. The figures in the table were obtained by mixing tubercle bacilli from a growing culture with the fluids, taking out samples from time to time, and sowing them on culture media. The values given may be regarded as being good for most sporeless bacteria, the sensitiveness of which is illustrated by this example. III. LETHAL VALUE FOR ANTHRAX SPORES. From Paul n. Kronig (55). 5% 30 seconds 1% I minute • • • 5 minutes i% 5 „ 10 „ 0-1% 10 o-3% 3 hours 0-25% 6 „ Substance. Corrosive sublimate Concentration. Time (Temperature 18° C.)- 7 % ( 16 litres) 12-14 minutes N itrate of silver 99 • Sulphate of copper . Sugar of lead . Sulphuric acid Caustic potash Permanganate of potash Bichromate of potash 0-84% 0-42% 0-2 % o-i % 4-25% 0-08% 1 6- % 32-5 % 4-9 % 5-6 % 3-95% ( 32 ( 64 (128 (256 ( 4 (200 ( i ( i ( ( ( ( 2 I 4 4 24-30 „ 45-60 „ 60-80 „ more than 120 minutes 15-16 minutes not in ioi hours not in io£ days not in 7 ,, not in 30 hours 1 8 hours 40 minutes not in 4 days 5 minutes 2 „ 2 „ not in 24 hours 120 minutes 7-4 % Permang. of potash, 8 litres, plus 8 litres HC1 Chlorine water . . 0-22% ( 32 litres) Bromine water . . 0-5 % ( 32 „ ) Carbolic acid . . 5 % • • • Formaldehyde . . 5 % • • • The lethal concentration for spores is determined in the following way. A suspension of the spores in water is dried upon silk threads, pieces of glass, or, better still, upon carefully-cleaned garnets, which are then laid in the disinfectant. Specimens are taken out at intervals and sowed in nutri- G 2 84 CHEMOTAXIS AND CHEMICAL DISINFECTION tive media. Before this is done, however, the threads or garnets, as the case may be, must be freed from any adherent disinfectant. Washing with distilled water is not enough for this, they must be rinsed with ammonium sulphide, and the soluble metallic salts precipitated and rendered harmless. Only in this way is it possible to obtain reliable results, for the small quantity of disinfectant adherent to the spores, although not sufficient to kill them under ordinary circumstances, might do so during the stage of imbibition, and would certainly destroy the germinating rod, thus giving a false idea of the disinfecting power of the substance. In the examples given in Table III, from 15,000 to 20,000 spores were killed in the stated times. The concentration is given in per cents., and also in terms of 'molecular dilution' ; for instance, 'Corrosive Sublimate, 16 litres,' means that sixteen litres of the solution contain the molecular weight of HgCl2 (i.e. 271) expressed in grams, so that 100 c.c. of the solution contains flff grams = 1-7 grams. This method of calculation, so much used in modern chemistry, is given because it permits an easy comparison of different salts— a comparison of molecule with molecule, so to speak. According to Koch absolute alcohol, concentrated glycerine, concentrated NaCl solution, and distilled water do not destroy anthrax spores, even after acting on them for months. The table shows that the halogen elements (chlorine, bromine, iodine) are the most powerful, and, among the metallic salts, corrosive sublimate. Nitrate of silver has some little effect, but sulphate of copper and acetate of lead are powerless. Free acids or free alkalies must be very strong to act as disinfectants. Chromate of potash, too, although it is a powerful oxidizing agent, has little germicidal power, whilst potassic permanganate, far less effective as an oxidizer, is a powerful disinfectant. Tables II and III show the great difference between spores and vegeta- tive cells in their behaviour to disinfectants. Five per cent, carbolic acid, for instance, which kills tubercle bacilli in thirty seconds, does not destroy anthrax spores in twenty-four hours ; with o-i per cent, sublimate, the times are ten minutes and sixty or eighty minutes respectively. Absolute alcohol, powerless against anthrax spores, kills tubercle bacilli in five minutes. The resistant power of the spores depends, no doubt, principally on the great im- permeability of the spore wall to dissolved substances of all kinds. This property is shared by the cysts and membranous coatings of all low organisms in the resting-stage, and by the seeds of plants. A resting-stage of long duration is indeed inconceivable without some such protection. In plant seeds and algal spores the impermeability is caused by the deposition in the membrane of fats and resinous bodies, and possibly the spore wall in bacteria is protected in the same way. Another cause of the durability of spores is the density of the protoplasm, which contains less water than it does in the vegetative cell. The great efficacy of corrosive sublimate as a disinfectant seems at first DISSOCIATION OF SALTS 85 sight to be only a special case of the toxicity of all mercury salts, and formerly it was thought that they were equally poisonous if only equal amounts of the metal were present. Equimolecular solutions therefore were supposed to be necessarily of equal disinfectant power. Recent investigations, however (55), based upon the modern theory of solutions, have proved this to be wrong, and rendered it probable that the toxicity of a poisonous salt varies with the degree of dissociation. The dissociation theory (56) has shown that in a solution of a salt there are present not only the unaltered molecules of the dissolved substance, but also a certain number of disintegrated or ' dis- sociated ' molecules. In a solution of HgCl2, for instance, a certain propor- tion of the HgCl2 molecules are split up into their electrically active components or ' ions,' the positive metallic ions (kations) Hg, and the negative ions (anions) Cl. The degree of dissociation, that is to say, the proportion of dissociated molecules to unchanged Hg C12 molecules, changes with the concentration of the solution, the temperature, and other conditions, and different salts of the same metal are dissociated in different degrees. Now many of the physical properties of a solu- tion, such as electrical conductivity, freezing-point, boiling-point, and osmotic pressure, depend upon the degree of dissociation, and it seems very probable that toxicity does also. Since different salts of mercury are very differently dissociated in watery solutions, we might expect that their toxic powers would be different also, and as a matter of fact we find this to be the case. A sixteen-litre solution (1-58 per cent.) of mercury cyanide, a salt which is but very slightly dissociated, does not kill staphylococci in three minutes, whilst a solution of corrosive sublimate only a quarter the strength (i. e. sixty-four litres, 0-4 per cent.) does. Anthrax spores left for twenty minutes in this solution of sublimate were almost all killed (seven colonies grew), whilst after a sixteen-litre solution of the cyanide, acting for the same time, innumerable colonies sprang up. A comparison of differently dissociated mercury salts shows clearly, therefore, the relation between dissociation and toxicity. The connexion becomes more striking still when we compare the same salt in different degrees of dissociation. The proportion of the dissociated molecules to the unaltered molecules in a given solution of a salt is constant. For example in a solution of HgCL the proportion of Cl ions to unchanged Hg C12 molecules is constant. By adding other chlorine ions, for instance by adding the more strongly disso- sociated NaCl, the number of dissociated Hg C12 molecules can be reduced, the reduction being dependent on the ratio between the amounts of disso- ciation of HgCl2 and of NaCl. Let us suppose a sixteen-litre solution of Hg C12 to contain x Cl ions and y unchanged molecules, then, broadly •j^ speaking, = c (a constant). If, now. there be added enough solid NaCl, 86 CHEMO TAXIS AND CHEMICAL DISINFECTION which is more readily dissociated than Hg C12, to make a sixteen-litre solu- tion, there will be present x + m Cl ions derived from the Na Cl. For the pure HgCl2 solution x = cy, but for the HgCl2 plus NaCl solution £*t _ ^ »/ x + (x + m] = cy or x — — -. In other words, the number of Cl ions 2 derived from the Hg Cl2 is reduced, its degree of dissociation is lessened, and some of the free Hg and Cl ions are built up again into complete molecules. The following table shows that as the degree of dissociation is lessened the toxicity is reduced. The figures give the numbers of colonies that arose from approximately equal quantities of spores after these had lain for six minutes in the solutions (55). Number of Colonies. 1 6 litre HgCl.2 solution .... 8 „ „ plus I NaCl . . 32 2 » » » 3 » • • » ;j )> 4 » • • 382 5j » » 4-6 „ . 410 j> » » 6 ,, 803 „ „ „ jo „ 1,087 The gradual diminution in germicidal power is unmistakable and needs no comment. These facts must be borne in mind in experiments on the disinfection, by sublimate, of liquids such as serum or bouillon which contain 0-7 percent. (eight litres) of NaCl. It is evident that more sublimate will be necessary than in fluids free from salt. The presence of peptone or albumen will necessitate a still further addition of sublimate, because some of it will form insoluble compounds with the proteids whereby its toxicity will be reduced. Since the degree of dissociation is dependent upon the nature of the solvent and also upon the temperature of the solution, these factors are of influence on the disinfecting power. The increase of toxicity which accom- panies increased temperature is not, however, solely the effect of augmented dissociation. Although the technique of disinfection has not as yet been affected by these recent discoveries, it does not need much penetration to see their high scientific interest and importance. There are many other bodies besides those mentioned that could be used for disinfecting purposes — anilin dyes, such as methyl violet, ethereal oils, and many aromatic compounds. New disinfectants spring up every day, and are widely advertised, only to disappear again very soon. Gases, such as carbonic acid, carbonic oxide, hydrogen, nitrous oxide, nitric oxide, sulphuretted hydrogen, sulphurous acid, and coal gas, arrest the growth of bacteria in agar cultures exposed to a slow stream of the gas, but the method has no practical value. The ozone in the air, too, DISINFECTION Ol' THE TISSUES 87 even where most abundant, is in such minute quantities as to be quite without influence. The fact that thousands of bacteria pass through our digestive organs every day suggests the question whether any of the digestive secretions function as natural disinfectants. The saliva and the pancreatic juice are alkaline in reaction and unable to injure bacteria. The pancreatic juice, by reason of the proteids it contains, is even nutritious. The bile acids inhibit the growth of bacteria, but of all the digestive secretions the free acid of the gastric juice (2-3 per thousand H Cl) alone is able to kill them. The action of this even is in any case uncertain, and can only affect sporeless cells. Normal gastric juice in a test tube destroys in half an hour the bacteria of cholera, typhoid and glanders, pus cocci, and the sporeless cells of anthrax and tetanus (57), but spores are not injured by passing through the stomach. To destroy anthrax spores, they must lie for six hours in 2 per cent. H Cl, so that it is evident the gastric secretion with only from -2 to •3 per cent. H Cl is quite inadequate, even if allowed to act for days. Even against sporeless bacteria it is of little effect, as the examples quoted show. Bacteria given to animals in their food (B. pyocyanens, blood in- fected with anthrax, tuberculous tissues) are not entirely destroyed even after six or eight hours (58). Chemical disinfection of the diseased body is not possible, for the tissue cells would be just as much injured by the disinfectant as the bacteria. Nor is it possible to disinfect by chemical means wounds in which bacteria have taken up their abode ; an antisepsis of wounds is not possible. When a wound cannot be purified by mechanical means the body must be helped to fight against the invaders, and the only way to do this is to secure clean- liness, asepsis. Asepsis confines itself to the treatment of wounds with germ-free instruments and dressings without using chemical disinfectants, and is sufficient to prevent clean fresh wounds, such as are made at operations, from becoming infected. As to the cause of the fatal action of disinfectants upon the bacteria we know but little. We know that the salts of the heavy metals, corrosive sublimate or nitrate of silver for instance, coagulate protoplasm. They destroy life probably by precipitating certain substances from the extremely complex protoplasm of the cells. Other compounds, such as alkalies and acids, may perhaps act by the separation and solution of proteid bodies, resulting in a destruction of the protoplasmic structure. Even a change in reaction might cause the precipitation of certain constituents. In most cases, however, the matter is beyond any explanation, because we have no knowledge of the peculiarities of protoplasmic structure on which the phenomena of life depend. CHAPTER X BACTERIA AND THE CIRCULATION OF NITROGEN IN NATURE 1. Introduction : The Assimilation of Free Nitrogen by the Bacteria of the Root Nodules of Leguminous Plants, and by Bacteria in the Soil. APART from the activity of organisms like the pigment and phos- phorescent bacteria, and the remarkable metabolism of the sulphur- and iron-bacteria, the work of bacteria in nature embraces three great processes : 1. The circulation of nitrogen: effected by putrefaction, the formation of nitrates, and the assimilation of atmospheric nitrogen. 2. The circulation of carbon by the, 'fermentation ' of carbohydrates and other non- nitrogenous products of animals and plants. 3. The causation of disease in other organisms, particularly in man and the higher animals. There are in nature five sources of nitrogen open to plants and animals : 1. The atmosphere (79 per cent, by volume of free nitrogen). 2. The nitrates of the soil and the traces of nitrous acid formed in the air during thunderstorms. 3. Ammonia, which occurs in minute quantities in the air, and is set free abundantly by the putrefaction and decay of dead organisms. 4. Animal excreta, which contain nitrogen compounds of many kinds, even down to ammonia ; and 5. The tissues of plants and animals. The first three of the above-named sources are useless to animals, for these obtain their nitrogen from plants only, either directly (herbivora), or indirectly through other animals (carnivora). To plants, on the other hand, nitrogen seemed until comparatively recently to be available only in one of these three forms. Vegetable physiologists had come to the conclusion that in nature the nitrates of the soil were the one and only form in which nitrogen was taken up by plants. For, although it was known that in experiments plants could be got to take up ammonia salts and even gaseous ammonia, it was evident that ammonia was not a common source of nitrogen in nature. And, finally, the atmosphere, the greatest storehouse of nitrogen in nature, seemed closed to plants. These views, however, were overturned by oy I-*RI-:E NITROGEX 89 detailed investigations into the nutrition of the Leguminosae. That the Legu- minosac could grow in soil poor in nitrogen and thrive thereon, even without nitrogenous manuring, had long been known, and it has now been demon- strated that they take up nitrogen from the air (50) and convey it to the soil. This enrichment is particularly evident when the plants are ploughed in. All other plants, all our cereals and food crops, are, as regards the soil, merely consumers of nitrogen, since they are unable to take it up in any form but that of the nitrates of the soil. The difference is seen even in the amount of nitrogen present in the tissues of the two groups of plants. Lupine seeds contain 5-7 per cent. N, wheat grains only 2-1 per cent., lupine haulms 0-94 per cent., wheat straw only 0-5 per cent. An experiment with peas showed that a quantity containing J 6 mgr. of nitrogen gave rise to a crop containing 499 mgr., whilst the quantity of nitrogen in the 4 kilograms of mould they grew in had increased from 22 to 57 milligrams ; a total gain of 518 mgr. of nitrogen. Calculated for larger quantities of plants it will be at once seen that these figures represent an enormous profit. One hectare ( = 2-4 acres) of lupines is cal- culated to enrich the earth by 227 kilograms of nitrogen annually. The amount gained by the meteoric fixation of nitrogen (production of HNCX and HNO3 during thunderstorms) would for the same area be only 0-09 to i -8 kilogram per annum, so that there is no doubt that it is the atmosphere, and the atmosphere alone, from which these plants extract the gas. Seeing that no other cultivated plants* (not even Brassica alba> the White Mustard) are able to act in this way, we apparently have in the Legu- minosae a remarkable and unique group of organisms. But we shall be wrong if we attribute the power of fixing free nitrogen to the tissues of the plant itself. They are powerless to do this, and behave towards nitrogen in no way differently from the tissues of non-leguminous plants. Not until they enter into partnership with certain bacteria, the bacteria of the root- nodules, do the plants act as usurers of nitrogen, gathering and storing it and growing ever richer. The nodules (60) which arise upon the roots of the seedling plants when they are a few weeks old are minute white or pinkish excrescences that soon increase in size and sometimes give the roots a distorted appearance, as though they were attacked by galls (Fig. 19, a and b). At first hard and smooth, they become wrinkled as the foliage of the plant grows and, by the time the pods arc ripe, are shrivelled and cracked. Their dried remains rot in the ground with the rest of the root. The nodules are either separate excrescences on the surface of the root, with whose vessels they arc connected by a tiny strand of vascular fibres, or the root itself swells up in places. In both cases there is always a close connexion between the cells of the nodule and the conductive tissue of the * The nodule-bearing alder and Elaeagnus are perhaps exceptions. 9o BACTERIA AND THE NITROGEN CYCLE plant (Fig. 19,^). If a transverse section of a young and firm nodule, which exudes a turbid milky juice on crushing, be examined, masses of large cells are seen, distinguished from those that surround them by being filled with a finely striate or punctate mass. Sometimes there are numerous small nests of cells, sometimes they run together to form larger masses. These cells, which constitute what was formerly known as the ^bacteroidal tiss e' (Fig. 19,$ and c], are nothing more or less than the enlarged cells of the root itself crammed full of fine, slender, rod-shaped bodies (Fig. 19 d). The nature of these has been variously inter- preted. Woronin, the first observer (1866), took them to be bacteria-like parasites ; other investigators since then have looked upon them as pro- teid crystals. If this view were correct we should have to regard the root- nodules not as pathological structures, but rather as a kind of nitrogenous potato with the albumen in the form of bacteroids. Recent investigations have, however, placed it beyond dis- pute that these rod-shaped bodies are bacteria, living and growing in the cells of the root (Fig. 19, c-f). The normal straight, rod-shaped bacteria, well tingible with anilin colours, are only found in the young nodules. As these grow older the bacteria take on all manner of distorted shapes ; spindle-shaped, branched, bifurcated or inflated forms are common. It is to these deformed bacteria only (Fig. 19, e and/) that the term bacteroids is still applied. They are so-called 'involu- tion forms,' similar to those found among many other species of bacteria when the conditions of growth are unfavourable (the acetic ferment, for instance, or the bacilli in old tubercle or diphtheria cultures). The cell contents as well as the shape are affected. They stain badly, and large numbers of the bacteroids seem to be mere empty shells. The conversion of the bacteria into bacteroids is, in short, nothing but a sign that the micro-organisms are dying and yielding up their protoplasmic contents to the plant, which begins to grow more vigorously as soon as the bacteroids appear. By the time the seed of the plant is ripe the shrunken empty nodules contain only fragments FlG. :Q. Root-nodules of Leguminosae. a, root nodule of the lupine, nat. size (from Woronin) ; 6, longitudinal section through a lupine root and nodule ; jf, fibro-vascular bundle of root giving off fine branches to every part of the nodule, and its bacteroidal tissue (w), (low power, from Woronin) ; c, a cell from a lupine nodule filled with bacteria (black) between which the finer stroma of the protoplasm is visible ; at the angles of the cells intercellular spaces ; from a section (fixed with Fleming's solution, stained by Gram's method). d, bacteria from root nodule of lupine, normal un- degenerate form ; e andyj bacteroids from Vicia villosa and Lupinus albits (from Morck). Magn. c 600, d-f about 1500. ROOT-NODULES OF LEGUMINOSAE of the bacteroids, together with a few intact healthy rods that remain in the soil and serve as seed material for next year's nodules. Root-nodules have been found in all the sub-orders of the Leguminosae (Papilionaceae, Mimoseac, Caesalpineae), and they always contain bacteria and bacteroids. Root-nodules without bacteria do not exist, and since Legu- minosae destitute of nodules behave with regard to nitrogen just as other plants do, it seems evident that the bacteria arc the nitrogen collectors. As an unproved hypothesis this idea was floating in the air for a long time, until the classical researches of Hcllriegel and Wilfahrt (59) placed the matter beyond the reach of controversy. Their experiments proved that it was possible by planting sterilized seeds in sterilized earth to grow leguminous plants for some months without root-nodules, if they were well protected from subsequent infection. They showed further that such plants had lost the power of storing nitrogen, but regained it if the earth they grew in were inocu- lated with an infusion of soil whereon Leguminosae had previously flourished. Finally their researches brought to light the marked contrast between nitrogen-storers like Leguminosae, and nitrogen-consumers such as wheat or oats. The following table gives a few of the chief results of their investigations. Amount of Nitrogen in seed and soil. Amount of Nitrogen in crop. Gain or Loss of Nitrogen in crop. I. NOT STERILIZED AND NOT INOCULATED: (a) Without nitrogenous manure Grammes. Grammes. Grammes. Oats 0-027 0-007 - O-O20 1 t\t"5 •••••• 0-041 1-283 + 1-242 (b) Manured with nitrate of calcium (N =0-112 grammes) Oats 0-139 0-09 - 0-049 Peas °-i53 0-700 + 0-547 II. INOCULATED WITH SOIL IN WHICH LEGU- MINOSAE HAD BEEN GROWN, NOT STERILIZED: (a) Without nitrogenous manure Oats 0027 0-007 - O-O20 Peas 0-038 0-459 -fO-42I (b) With nitrate of calcium (N=o-ii2 grammes) Oats 0139 0-088 - 0-05I Peas 0-150 O-22O -f 0-070 Ill . INOCULATED AND STERILIZED: (a) Without nitrogenous manure Peas 0-038 0-015 - 0-023 (b) With nitrate of calcium (N = 0-1 1 2 grammes) Peas 0-045 0-014 -0-03I 92 BACTERIA AND THE NITROGEN CYCLE These tables explain themselves. It is clear that the pea nourishes itself in a similar manner to the oat when cultivated in sterilized soil and unable to form nodules (III), whereas under natural conditions it bears these prolifically and stores up nitrogen in large quantities. Furthermore, the tables show that, whilst oats are rendered much richer in nitrogen by suitable manuring (I b and II £), peas are not affected (III); they cannot utilize nitrogen offered to them in this form. For oats, on the other hand, it is a matter of indifference whether the soil is sterilized or not, since they form no nodules. The advantage gained from non-nitrogenous manure, such as phosphate of potash, is shared equally by oats and peas. The next task was to obtain pure cultures of the bacteria of the root- nodules, and ascertain their behaviour towards atmospheric nitrogen. They are easy to cultivate on a decoction of leguminous plants to which \ per cent, asparagin and 2 per cent, sugar has been added. In such a medium the bacteria grow well in the form of slender rods, aerobic and mobile, with a tendency to produce involution forms or bacteroids. They at first get their nitrogen from the asparagin, but after about two months' growth every litre of the culture has gained from 9 to 18 milligrams of nitrogen which must necessarily have come from the atmosphere (Beycrinck). Maze in similar experiments obtained an increase of 47-5 mgr. nitrogen in fifteen days, and in another case 23-4 mgr. in eighteen days, so that although further investigations are desirable there can be no doubt that pure cultures of the bacteria from root-nodules do actually assimilate and fix the nitrogen of the air (61). Morphologically the bacteria from different kinds of Leguminosae, whether in the nodules themselves or in pure culture, are very similar in appearance, and the growths on gelatine (which is not liquefied) are all of the same character. The ' bacteroid ' forms are likewise of the same kind in all cases, so that it would seem as if all Leguminosae were inhabited by the same species of bacterium, to which the name Bacillus radicicola was given by Beyerinck and RJiizobinni leguminosarum by Frank. But as soon as we investigate the action of these bacteria upon plants we find differences between them. If plants of clover, pea, and vetch growing in sterilized earth and destitute of root-nodules be watered with an infusion of bacteria derived from the root-nodules of peas, we find that numerous root-nodules arise both on the pea and on the vetch, whilst the clover forms very few or none at all. Conversely, clover bacteria are almost useless to the pea and the vetch. Nobbe and Hiltner (62) as a result of experiments, which although successful are not beyond criticism, have come to the conclusion that it is only between members of the same group of Papilionaceae that the bacteria of the root-nodules are exchangeable, that, for instance, clover bacteria can be used by other Trifolieae such as lucerne, but not by Phaseolus lupines S } 'MB /OS IS : PA RAS1 TISM 93 or other Phascoleae or by Viciae such as Vicia, Ervuni, or Pisum, the bacteria of these again being useless to Trifolicae. We should have, in fact, if these views be correct, different breeds of one and the same species of bacterium (D. radicicohi), comparable to the different races of brewers' yeasts or to those described by Eriksson in the wheat rust-fungus Puccinia. Nobbe and Hiltner have attempted to turn their improved theories to practical account by the introduction of ' Nitragin' a preparation manufac- tured at the Hochst Chemical Works. Nitragin is a pure culture on gelatine of bacteria from root-nodules — a kind of living manure to be mixed with the seed material or soil and strewn over the fields. There are eight different sorts of nitragin on the market, suitable for peas, lupines, beans, &c., and it is said to be very advantageous when first planting Leguminosae in virgin soil, or in soil of poor quality, moors for instance, or where for many years no Leguminosae have grown and the land is presumably destitute of nodule organisms. The results are extremely inconstant and frequently very difficult to judge, so that it is not surprising that opinions are divided as to its efficacy *. Instead of nitragin, 'leguminous earth,' i.e. soil in which Leguminosae thrive well, is sometimes used with advantage to ' inocu- late ' new ground and bad earth such as moorland. The remarkable association of bacteria with leguminous plants is generally re- garded as an instance of symbiosis, a connexion from which both parties reap advantage similar to the ' mutual ' associa- tion of alga and fungus in lichens. Here, as is well known, the green or brown cells of the alga are enclosed by the thickly matted filaments of the fungus (Fig. 20). The alga supplies the metatrophic fungus with organic compounds and the fungus repays the debt by providing the alga with the necessary moisture and mineral salts, not to speak of the protection that its embrace involves. This at least is the opinion of those who, following the ' symbiotic ' tendency of the day, look upon a lichen as a co-operative concern. But it is more than doubtful whether this view is correct. All the algae that live in lichens are known to be able to live in a free state. They certainly do not need to be supplied by the fungus with water and mineral salts, and as regards protection it is difficult to see how the algal cells can be ' protected ' by hyphae which cling round them tightly and send ' suckers ' into their interior (Fig. 20, b). The fungus behaves in every way as a parasite which lives upon FlG. 20. Parasitism of Lichens, a. section through the thallus of Xanthoria parietitia (from Sch wendener) ; 4, algal cells surrounded by fine fungous hyphae in Cladonia furca/a (from Hornet). The green algae dotted black. Magn. a 500, b 950. * [See Dawson, /'////. Trans. 1899, Vol. 192, pp. 1-28.] 94 BACTERIA AND THE NITROGEN CYCLE the alga. As a parasite it must establish a close connexion between its tissues and those of its host, and, since it cannot crawl into the alga as the tape-worm does into man, it winds its hyphae around the cells. Enclosed thus in the light, well-ventilated thallus of the fungus, the algae continue to live, but they draw no nourishment from it. The advantage is entirely on the side of the fungus, which is a true parasite. The association between the leguminous plants and the bacteria of their root-nodules, paradoxical as it appears at first sight, is of a precisely similar character ; the plant is parasitic upon the micro-organism. To render this view more comprehensible, we will consider in detail the development of the nodules. The fine root-hairs of a young leguminous plant, destitute as yet of nodules, push themselves everywhere into the crannies between the particles of soil, taking up water and mineral salts and secreting fluids which dissolve and render assimilable the substances with which the cell-wall comes into contact. There can be no doubt that some of the secretions of the root-hairs act chemotactically upon many of the bacteria that swim in the water-filled crevices of the soil ; and these would be attracted furthermore by organic sub- stances issuing from \vounds or scratches upon the root-hairs or upon the epi- dermis of the root. Asparagin, one of the most powerfully chemotactic com- pounds known, is present in large quantities in the tissues of germinating plants, and must necessarily escape wherever the cell-walls are injured. It is quite possible that this substance acts as a ' bait ' to attract the bacteria which would swarm into a torn root- hair just as they do into a capillary filled with asparagin (Fig. 21, b). There are reasons, too, for thinking that the plant facilitates the entrance of the micro-organisms by a softening of the cell-membrane of the root-hairs. This much is certain, that the bacteria are chemotactically attracted to the plant, and that, nourished by the organic substances offered to them, they multiply rapidly and force their way from the surface of the root inwards. Here again by softening its cell-walls the plant seems to smooth their path, and in thick columns the bacteria zoogloea* pushes on from cell to cell * The so-called ' infection-thread,' first observed penetrating the root-hairs by Marshall Ward (see Phil. Trans. 1887, Vol. 178, p. 545). FIG. 21. Invasion of leguminous roots by bacteria. a, Cell from the integument of root of the pea with nucleus and so-called 'infection thread," a broad stream of bacteria zoogloea that pushes its way through the cell walls (from Prazmowski) ; 6, end of a root-hair of the pea ; at the right, particles of earth, and on the left a mass of bacteria have gathered. Inside the hair, protoplasm mixed with bacteria which are pushing their way in a thin stream upward. (From B. Frank.) BIOLOGY OF THE ROOT-NODULES 95 into the inner tissues of the root (Fig. 21, a & b}. And now the plant begins visibly to react to the stimulus. Many of the cells of the root become larger, and carbohydrates and asparagin are brought down from the upper parts of the plant to be given unstintingly to the micro-organisms, whose accelerated growth and multiplication soon become manifest in the fast developing nodules. The root-nodules are nothing else than pure cultures of bacteria maintained by the plant for its own ends. The microbes, which grow at first at the expense of the tissues they live in, become before long more independent and draw their nitrogen from the atmosphere. Carbon they most likely obtain the whole summer through from the plant, which probably also secretes the ferment by which the starch is changed into assimilable sugar. The root-nodules are now in full activity, the bacteria in the cells absorbing nitrogen from the air in the intercellular spaces (63), and storing it up in their own bodies. But this state of exuberant vitality does not last. From overcrowding and other causes the multiplication of the bacteria becomes less rapid and bacteroids (involution forms) appear, a sign that the conditions of life are becoming less favourable. The plant asserts itself, and begins to absorb the substance of the bacterial cells, conveying the nitrogen from the root-nodules into the growing seeds. In the lupine at the time of flowering the nodules contain 5-2 per cent. N, which then becomes gradually less until, by the time the pods are ripe, only 1-7 per cent, is present. It is the nodules alone which yield up their nitrogen in this way, the amount contained in the other parts of tJie root remaining from first to last unchanged, namely about 1-6 per cent. The precise way in which the leguminous plant absorbs the bacterial cells is not clearly understood, but in all probability it dissolves them by means of a peptonizing enzyme. Only a remnant of the bacteria return to the soil when the root decays, the greater number are literally eaten up by the plant. To call this symbiosis is certainly a misapplication of the term *. The carbohydrates and asparagin that the plant offers the bacteria in the beginning are nothing but a usurer's loan, for in the shape of valuable nitrogen they are demanded back again later on with heavy interest. Looked at from this standpoint, the view that the leguminous plant is parasitic on the bacteria no longer appears absurd. It is obliged to ' put itself outside ' its hosts, just as the fungus is obliged to enclose the algae in the lichen thallus. But, whilst the lichen fungus is completely parasitic, the leguminous plant is only partly so. It only derives its nitrogen from its host, supplying its other needs (carbohydrates, mineral salts, &c.) in the same way as all ordinary plants, from which it differs indeed only in being unable * [It should be pointed out that the author's views of the relations between the organisms here and in the Lichens are not those accepted by botanists generally. See, for example, PfefTer, Physiology of Plants, Vol. I, Engl. ed., pp. 364 and 371.] 96 BACTERIA AND THE NITROGEN CYCLE to assimilate nitrogen from the nitrates in the soil. The table (III) on p. 91 shows this very clearly. In this semi-parasitic mode of life the Leguminosae resemble the Rhinanthaceae, Thesinm, and some other green plants, of which the roots graft themselves on to the roots of adjacent plants of other species. These function as hosts, but it is not known what substances they yield up to their parasites. Since it is evident that the soil of every field in which Leguminosae grow, and probably indeed soil of all kinds, contains the bacteria of root- nodules, attempts have naturally been made to obtain them direct from the soil. Up till now these experiments have not been attended with success, nor is it known whether the bacteria live and multiply in the soil, or whether they exist merely in the form of spores (as yet unknown) which are quickened into life by contact with the roots of leguminous plants. But although it has been hitherto impossible to isolate the bacteria of root-nodules from soil, Winogradsky has discovered another soil bacterium which fixes atmospheric nitrogen (64). It has been named Clostridium Pasteuriamtm, and belongs to the group of butyric ferments. It can be isolated in a nutritive solution containing sugar (as a source of carbon) and mineral salts, nitrates or other nitrogen compounds being of course rigidly excluded. The sugar is broken up into butyric, acetic, and carbonic acid, and hydrogen, besides some by-products of uncertain composition, and at the same time nitrogen is taken from the atmosphere and stored up in the body of the cells. The more sugar there is present (i.e. the more energetic the fermentation), the greater is the amount of nitrogen taken up, as the following figures show : Dextrose in nutritive N. in nutritive N. in the crop solution. solution. after fermentation. Grammes. Milligrammes. 1 o 3.0 2 O 2-9 3 o 8-1 6 o 12-8 Possibly the nitrogen is fixed by nascent hydrogen, and ammonia formed as the first product. The bacterium in pure culture is an anaerobic actively motile bacillus \vhich grows in slimy masses. It is considerably different in appearance from the bacteria of root-nodules, becoming spindle- shaped during sporulation (hence the name Clostridium) and, like other butyric bacteria, it stains blue with iodine. What the conditions are under which Clostridium Pasteuriamim exists in nature, and whence it obtains its carbon, is unknown. Should sugar be a necessity it could not grow in unmanured soil, but might occur in situa- tions where the processes of fermentation and putrefaction give rise to a variety of organic compounds. NITROGEN-FIXING BACTERIA 97 It is not known whether other butyric organisms arc also able to fix atmospheric nitrogen, but presumably other, perhaps prototrophic, bacteria of the soil do so. It is highly probable that we shall find such in forests where the earth is never ' manured ' and where, notwithstanding, enormous quantities of nitrogen are fixed and worked up into the living substance of plants. But all researches in this direction must be received very critically, for there can be no doubt that the idea now very common in agricultural circles that all soil bacteria can assimilate free nitrogen is quite incorrect. Green and blue-green algae were formerly supposed to be able to do so, but this opinion is now held to be incorrect ((55), and although it is stated that mould-fungi can fix free nitrogen the experiments on which the state- ment is based did not take into consideration all the possible sources of error. The time will perhaps come when ' Nitragin ' will be replaced by pure cultures of free-living, nitrogen-fixing bacteria, which will be 'sown' so as to alternate with nitrogen- consuming crops. FISCHER II CHAPTER XI BACTERIA AND THE CIRCULATION OF NITROGEN (Continued] 2. The Liberation of Organic Nitrogen by Putrefaction, and its Mineralization by the Nitrifying Bacteria. \ ONE of the most conspicuous physiological differences between plants and animals is the absence in plants of any nitrogen-containing excretions. Nitrogen, once assimilated by the plant, is built up into proteids, alkaloids, colouring-matters (chloropJiyll, indigo), and other compounds, but as long as the plant lives is not excreted. It is set into circulation again only by the death and decay of the tissues, for the living plant can be used as a source of nitrogen only by parasites and by plant-eating animals. In the animal body nitrogen is present chiefly in the form of proteids and their derivatives the albuminoids, e. g. mucin, glutin, keratin, elastin, as well as other highly complex bodies such as lecithin, haemoglobin, chilin, and nuclein. In the animal, as in the plant, the nitrogen of these substances is set free at death,, but, in addition, the animal, unlike the plant, gives off in its secretions (milk) and excretions (dung, urine) a number of nitrogenous compounds. It is to these substances, enriched still more by the nitrogen contained in straw, that stable-manure owes its high value as a fertilizer for plants. But the nitrogen in fresh stable-manure is in a form that is useless to plants ; they cannot assimilate it. In the urine of herbivora nitrogen is present mainly as hippuric acid, in human urine mainly as urea and uric acid. In the excrements it is contained in the remnants of undigested nitrogenous food, and the products of decomposition set up by the bacteria of the intestine. These products include indol, skatol, leucin, tyrosin, and many simpler nitrogen compounds even down to ammonia. But none of these, not even ammonia, are directly available as nutritive materials for green plants. Not until the nitrogen has been removed from the organic molecule by the process of putrefaction, and has been united to a mineral base by the process of nitrification, can it be taken up by the green plant. PUTREFACTION 99 Through the tissues of green plants all organic nitrogen must, however, sooner or later, circulate. Putrefaction is a purely biochemical process, and can only take place when the fundamental conditions for all vital action are fulfilled. If the temperature sink below a certain point, organic substances cannot putrefy, as was well shown by the frozen Siberian mammoths. When discovered, their flesh was so little changed that it was eaten by the hunters' dogs ; yet it must have lain in Nature's refrigators for countless centuries. Absence of moisture acts in the same way as absence of warmth. Dry meat does not putrefy. Dryness and a low temperature together will preserve organic matter for any length of time, as is well illustrated by the undccayed yet unembalmed bodies that have been kept for centuries in the vaults of certain churches. Other means of preservation by chemical and physical agencies have been already referred to, but in all methods the funda- mental principle is the same, namely, to create such conditions that bacteria cannot live ; for putrefaction — the splitting-up of the nitrogenous con- stituents of organic matter — is the work of bacteria, and of bacteria alone. The flesh of fruits, being poor in nitrogen and rich in organic acids, is not attacked by bacteria. The rotting of pears, grapes, oranges, and other fruits of a like character, is brought about principally by mould-fungi (66) (Penicilliinn, Mncor, Botrytis). The decomposition of dead animal bodies, of vegetable tissues, or of substances like stable-manure, is far from being a simple putrefactive process. Side by side with the disintegration of nitrogenous bodies there are going on a number of fermentative changes by which non-nitrogenous compounds are being broken up, besides nitrification and other biochemical processes. For this reason it is always difficult, and often impossible, to determine the respective parts played by the different species of bacteria. Putrefactive processes are going on in all places where nitrogenous organic matter is under suitable conditions of temperature and moisture, manure heaps for instance, cesspools, the muddy bottoms of lakes and rivers, and the ocean floor. Proteids are split up by putrefaction into a large number of simpler compounds both nitrogenous and non-nitrogenous. The substances thus produced are precisely similar to those resulting from the artificial decom- position of proteids by fusion with caustic potash, or boiling with hydro- chloric acid or barium hydrate. Five groups may be distinguished : 1. Albumoses and peptones : soluble diffusible bodies closely resembling albumen. They are produced by the action on albumen of bacterial enzymes similar to the enzymes (pepsin and pancreatin) which give rise to peptones in the digestive tract of man. 2. Aromatic compounds : among others indol and skatol which give the characteristic odour to human excrement ; also some non-nitrogenous substances such as phenol, phenylacetic acid, and phenylpropionic acid. 3. Amido lompoHiids, all nitrogenous; leucin, tyrosin, aspartic acid, glycocol. II 2 ioo BACTERIA AND THE NITROGEN CYCLE 4. Fatly and aromatic acids, all non-nitrogenous and therefore having no part in the circulation of nitrogen ; acetic, butyric, succinic, and valerianic acids. 5. Inorganic end-products of putrefaction : free nitrogen, ammonia, free hydrogen, methane, carbonic acid, methylmercaptan, sulphuretted hydrogen. It is probable also, but not certain, that phosphuretted hydrogen is formed and is oxidized at once by the free oxygen of the atmosphere. Most of these substances are formed also by the chemical decomposition of proteids, but there is a sixth group which may be termed specific putrefac- tive products. These are the so-called ptomaines or putrefactive alkaloids (67). They are nitrogenous compounds (amine-bases), but although a large number have been described they are, in consequence of the difficulty of preparing them, still only imperfectly known. From putrid flesh (mammalian, including human), fish, and gelatine Brieger isolated neuridine (C5 H14 N2), trimethylamine (C3H9N), cadaver ine (pentamcthylendiamine, Cr> H14 N2), and pntrescine, a diamine of the methylene series (C4H12N2). These are all of them either not poisonous or only poisonous in large doses. Other ptomaines, derived from putrid foods of various kinds (sausages, cheese), are highly toxic (Ptomatr opine, Tyrotoxine). The name toxine was formerly used for the amine bases only, but it is now applied indiscriminately to all bacterial poisons irrespectively of their chemical constitution. (See also Ch. XVII.) As far as the circulation of nitrogen is concerned, we have to consider only the end-products of putrefaction, free nitrogen and ammonia. All the more complex intermediate bodies are broken down finally to these. Leucin, for instance, splits up into valeric acid, ammonia, carbonic acid, and free hydrogen. Tyrosin yields by aerobic putrefaction hydroparacumaric acid, paraoxyphenylacetic acid, paracresol, phenol, ammonia, and carbonic acid. By anaerobic putrefaction it gives rise to indol, carbonic acid, and hydrogen. The phenomena of putrefaction are so complicated that we do not know all of the compounds that arise during the process. The course it runs is, however, greatly modified by the presence of oxygen (68). This list of putrefactive products is far from being complete, for even the qualitative investigation of the processes is still unfinished ; quantitative analyses are at present impossible. We do not know for instance what determines the predominance of one or the other intermediate product. The effects of the presence of oxygen are somewhat better understood (68). If air have free access, putrefaction may go on without any odour at all, the evil-smelling gases (NH3 and SH2 for example) being oxidized at once to form nitrates and sulphates. Aerobic bacteria too, such as the nitre and sulphur bacteria, bring about this mineralization of organic nitrogen. Moreover, when air is circulating freely, there is no accumulation of intermediate products such as skatol or indol. To this kind of aerobic decomposition, proceeding without offensive smell, the term ' decay ' may be applied as distinguished from ' putrefaction.' It occurs on the surface of manure-heaps, on the outer surface of carcases, and in well-ventilated soil. PUTREFACTION 101 In anaerobic decomposition (putrefaction proper), as in anaerobic fer- mentation, the organic molecules are at first only partly disintegrated, inter- mediate products such as leucinc, tyrosinc, skatol, and indol being formed. In the absence of air these accumulate, and hence it is that the putrefaction going on in the mud of ponds or ditches, or inside carcases, is accompanied by such evil odours. Although the details of the process vary considerably according to the presence or absence of air, the ultimate products of putrefaction are in both cases the same : namely, free nitrogen, free hydrogen, ammonia, methane, carbonic acid, and sulphuretted hydrogen. These also are the end-results of the disintegration of the human body. The decomposition of plant tissues, poor in nitrogen but containing large quantities of cellulose, gives rise to humin compounds. There can be no doubt that it is a biochemical process, but the nature and functions of the bacteria concerned in it have not yet been carefully studied (69). One of the bacteria which was formerly looked upon as a putrefactive organism par excellence was the Bacterium tcnno. Cohn described under this name a short, ovaL actively motile bacillus, slightly fluorescent in cultures (Fig. 22, a}. The species is, however, no longer to be identified, for his description would apply to several common forms. Asa matter of fact there are many bacteria going by this name which is properly only applicable as a collective designation for motile bacteria in putrescible substrata. In the rich bacterial flora of a putrescent fluid (70) two distinct biological groups must be distinguished, the saprogenic and the saprophile. To desig- nate all bacteria living upon dead organic matter as saprophytes is insuffi- cient. As we have already seen, the saprogenic bacteria are able, by the chemical ferments they secrete, to break up the molecules of proteids. The saprophile organisms cannot do this, but they find a rich supply of nutri- ment in the products thus formed. A large number of metatrophic species are endowed with this faculty, including some pathogenic forms (cholera and typhoid), and water bacteria like Spirillum nndnla. The sulphur bacteria too must be called saprophile where they form a coating over the rotting vegetation under water, and the nitrifying organisms also when they oxidize the NH3 evolved from decaying organic matter. The bacteria of the other division, the true saprogenic organisms, include very numerous species, some of which are much better known than others (e.g. Hauser's B. (Proteus vulgaris], but all of which possess the power of breaking up the complex protcid molecule more or less quickly into simpler groups. With the exception of certain toxines in a few cases, specific products which would serve as classificatory characters are not known. Indol, for example, the production of which was once thought to be characteristic of the cholera germ, is produced by all the cholera-like vibrios, as well as by the B. coli and other forms (Fig. 22, b}. Near to 102 BACTERIA AND THE NITROGEN CYCLE these pathological bacteria with saprogenic properties many of the fluorescent and photogenic bacteria would have their place. Bacillus fluorcsccns liqnc- faciens, an actively motile aquatic species, breaks down albumen into peptones, fatty acids, and other compounds. One of the bacteria of the intestine, described formerly as Bacillus putrificus coli, yields peptones, indol, skatol, amides, and ammonia. Similar to this species in its action on albumens are the toxine-forming B. vulgaris (Proteus vulgaris] and its allies. B. vnlgaris makes its appearance almost always in meat infusions exposed to the air. It is a slender rod 1-5 — 4111 long by 0-5 // thick, with a tendency to form chains. It is peritrichous and actively motile (Fig. 22). Closely related to it are a number of forms (Kurth's, B. Zopfii, and others), 9 FIG. 22. Putrefactive bacteria, a, Baclrillum psetidolermo, corresponds most closely to the old Ba&ertutK terino of Cohn. b, Cholera-like vibrio from putrid water, c, Bacillus ureae, the commonest cause ofurinous fermentation, probably identical with Pasteur's Micrococcus ureae. d-h, Bactridium Proteus {Bacillus Proteus, Bacterium Zopfii, Proteus inilgaris^ &c.). rf, peritrichous rod, cobweb-like growth (zoogloea) on gelatine (50 diam.) ; f, portion of same more highly magnified (300 diam.); g, tree-like growth on gelatine with thickenings; //, part of same magnified 300 diam. to show the structure of thickenings which consist of closely wound threads. Magn. a-d about 1500, e and ^50, _/~and A 300. all placed at one time by Hauser in one genus Proteus ; so called on account of the manifold shapes of their colonies on gelatine*. These colonies are ramified zoogloeae having under a low power much the appearance of fungus mycelia, covering the gelatine with a filamentous growth. Each filament consists of chains of bacteria united by a jelly-like substance (Fig. 22, d-Jt). These apparently specific putrefactive bacteria must not be looked upon as an exclusive group, such as the sulphur or nitrifying organisms, whose metabolism is of a circumscribed kind and which can only thrive when its peculiar conditions can be fulfilled. The various species of Proteus, for instance, have zymogenic as well as saprogenic properties. They can ferment carbohydrates, producing gas and acid during the process, as does also Bacillus coli commnnis. See Annals of Botany, Vol. xiii, 1899, p. 198, for an account of Proteus. /•'/•: A'.)/ i:.\r. i TK )N 103 Very careful chemical investigations on pure cultures will be necessary before the chaos of phenomena presented by the putrefactive bacteria can be arranged in something like order. In view of the present state of our knowledge of these organisms, it will be best perhaps to make the definition of a putrefactive bacterium rather wide, and include all bacteria with saprogenic properties, whether they possess other plcotrophic properties (e.g. zymogenic) or not. It is certain that many bacteria, almost all the cocci for instance, and many pigment bacteria, are destitute of saprogenic power. Saprogenic bacteria arc able to break up albuminous bodies of every kind and of every morphological character, whatever organs of the body they be derived from. The influence of these saprogenic properties in the production of disease will be considered later on. Just as the Bacterium tcnno was formerly regarded as the only cause of putrefaction, so was the Micrococcns urcae discovered by Pasteur regarded as the specific microbe of urinous fermentation. Healthy human urine, sterile and acid when passed, becomes sooner or later alkaline. The urea has been converted by addition of water into carbonate of ammonia : CO (NH2)2 + 2H2O = CO3 (NH4)2. A similar decomposition is undergone by the uric acid and by the hippuric acid in the urine of herbivores. It is caused by a bacterial ferment and, as with the putrefactive organisms, it was formerly thought that one species alone was at work. This was the Micrococcns rireae of Pasteur, a short, almost spherical, non-motile cell generally growing in pairs (Fig. 22) or little chains (71 and 112). We know now, however, that many different kinds of bacteria are able to cause the ammoniacal fermentation of urine. No less than sixty species are said to occur in manure and sewage, among others the B. vulgar is. On the other hand, B. subtilis, B. anthracis, B. typ/ii, V. cliolerac, the pus cocci, and many saprogenic forms arc unable to split up urea. Conversely, the urea bacteria are incapable of decomposing albu- minous bodies, which is not surprising, seeing how different the two processes are chemically. The resting cells of the urea bacteria are everywhere present in urine, manure, earth, air, and dust. All urine that is left exposed to the air is sooner or later attacked by them, and its nitrogenous constituents arc converted into ammonium carbonate. In this way the path is prepared for the nitrifying organisms, by which the nitrogen is made ready for renewed circulation through the tissues of green plants. The amount of nitrogen treated in this way by bacteria is enormous. The sewers of Leipzig discharge every day, in the form of urine, no less than 8,400 Ib. of nitrogen. The two great processes, putrefaction and ammoniacal fermentation, are going on in nature uninterruptedly. In the soil, in the mud of lakes and 104 BACTERIA AND THE NITROGEN CYCLE rivers, in all the sewers and refuse heaps of human communities, the two classes of micro-organisms are incessantly at work, sometimes alone, some- times together, and the greater part of all organic nitrogen is sooner or later liberated in the form of ammonia, together with a small amount of free nitrogen*. The free nitrogen is at once utilisable by the root-nodule organisms, and by other bacteria in the soil. The ammonia nitrogen, on the other hand, must be ' mineralized ' before it can be used by green plants, must be oxidized and combined with a base to form a nitric salt f. This change, known as nitrification, was formerly thought to be a purely chemical phenomenon, the oxidation being presumably effected by the free oxygen of the atmosphere. This view is now known to be incorrect, evidence having gradually accumulated which proves irrefutably the biochemical nature of the process. The bacteria to whose activity it is due defied for many years all attempts at isolation, until Winogradsky, the Russian in- vestigator, succeeded in separating them from the soil and obtaining pure cultures (72). His researches too brought to light the unique fact that the nitrifying bacteria are absolutely prototrophic in their mode of life — and we owe to him glimpses into the conditions of existence among the lowest organisms. The nitrifying bacteria are everywhere present in the soil of our gardens, fields and meadows, and in the virgin earth of the mountain sides and plains, untiringly at work preparing the food for plants. They have been cultivated by us unconsciously for centuries in the ' saltpetre beds,' where putrescible material (manure and other animal remains — skins, horns, glue &c.) is mixed with lime and allowed to ferment, and there can be little doubt that the Chilian nitre owes its origin to the action of nitrifying bacteria in a recent geological period. It probably arose from the washing down of saltpetre that had accumulated around putrefying organisms in the rainless regions of the coast. The isolation of the nitrifying bacteria is not difficult ; but rather different methods are necessary from those used for other bacteria. Highly nutritious culture media are useless, for the nitrifying bacteria are proto- trophic in the strictest sense of the word. They may be cultivated en masse by inoculating with a little garden earth the following solution : Grammes. Water ....... 1000- Potassium phosphate (dibasic) . . «2 Magnesium sulphate .... «3 Soda (or MgCO3) .... «5 Sodium chloride «5 * The nitrogen of crop residues and of the ploughed-in fallow undergoes just the same changes, t This applies of course also to the sulphate of ammonia from gas-works, so largely used as manure. NITRATE AND NITRITE BACTERIA 105 To these must be added from 20 to 50 mgr. of sulphate of ammonia that should be renewed in larger doses (i gr.) at intervals of about a week. The only source of nitrogen is the sulphate of ammonia ; the carbon is obtained from the CO.3 of the atmosphere, not from the sodium or mag- nesium carbonate, which are added only to neutralize the nitric and nitrous acid that arise from the activity of the bacteria. The sodium chloride benefits them in some way at present unknown. The nitrogen of the ammonia is not converted directly into nitric acid, as was formerly supposed, but oxidized first of all to nitrous acid and then to nitric. There ..arc two distinct reactions going on, the formation of nitrites from ammonia, and of nitrates from nitrites. Each is performed by specific bacteria, which are always present together, so that the compounds formed by the one kind are immediately taken up by the other and the end-products alone appear in the soil. Only in pure cultures is it possible to follow each process separately. Both are very slow ; in a sixteen days' old culture, 60 mgr. of (NH4)2SO4 were converted into nitrous acid daily, and in a six weeks' old culture 64 mgr. potassium nitrite daily into nitric acid. Probably, under natural conditions, the process is more rapid. To isolate the nitrite from the nitrate bacteria, Winogradsky used the customary A methods of plate culture, substituting colloid » \ silica for the gelatine *. Very thoroughly washed agar may also be used. For the nitrite organism (NH4)2SO4 is added to the medium, for the nitrate bacteria KNO2. F,G. „. Nitrifying bacteria TM *i_ «i_ t_ • 1- • i i i TIT- praciskv). a, Nitrosomotias ei/i'oftea. a 1 he nitrite bacteria are divided by VVino- nitrite bacterium from Zurich. /,, Witroso- i i i • i . , JIT-.. monas jaz>anensis, a similar organism from gradsky mtO tWO biological genera, NltrOSO- Java. *, Nttrobacter, a nitrate bacterium i -\T. TVT-J • from Quito. Maen. 1000. coccus and JMitrosomonas. Nitrosococcus is a non-motile spherical cell (^ju. in diameter, and less) found in soil from South America and Australia. Nitrosomonas is a very short ellipsoidal, motile form, of which two species in particular may be mentioned. Nitrosomonas curopea (Fig. 23, a), ubiquitous in the soil in Europe, Africa, and Japan, is from 0-9/0. to i // wide, and i-2/z to i-8/x long, with a short cilium. N. javanensis from Buitenzorg (Fig. 23, b] is almost spherical (0-5-0-6 JM), with a cilium 30 \i long. This is the longest cilium known among bacteria. Spores have not been seen. The nitrite bacteria render the culture turbid as long as they arc motile. They also form zoogloeae, and if crystals of Mg C O^ be added the bacteria collect on them in slimy masses and eat into the mineral just as lichens eat into a stone. * For details respecting the composition of the media, see works cited in Notes (pp. 169-184). 106 BACTERIA AND THE NITROGEN CYCLE The nitrate bacteria (Nitrobacter, Fig. 23, c] are minute non-motile rods (0-5 ju, by 0-25 ju) that grow without causing turbidity in the medium. They form delicate pellicles on the floor and sides of the culture glass. Spores are unknown. As might be expected in the case of organisms with oxidizing functions, all the nitrifying bacteria are aerobic. They require no light, and yet in spite of this are able to assimilate the CO2 of the atmosphere. In three experiments, where the nutritive solution contained originally 6 mgr. CO2 in the form of MgCO3, the cultures after seven weeks' growth showed 37-6 mgr., 26 mgr., and 17-5 mgr. CO2 respectively. This was subsequently proved by Godlcwski (73) to have been absorbed entirely from the air without light and without chlorophyll. As already mentioned, the nitrogen was derived from the ammonia, or from the nitrous acid. A little nitrogen is even set free. The energy necessary for the processes of life is gained by the nitrifying bacteria from the oxidation of the ammonia, or the nitrous acid. The materials from which they build up their cells are therefore inorganic compounds of the very simplest character, carbon dioxide and ammonia or nitrous acid, with a few mineral salts. They are thus protolrophic in the strictest sense of the word, for a simpler synthesis of proteids than theirs is scarcely conceivable. Facts have recently come to light which show that green plants do not get the full benefit of all the nitrates formed in the soil by the nitrify- ing bacteria (74). Nitrate-reducing bacteria have been discovered which destroy that which the nitrifiers have built up. Several species of these denitrifying organisms have been isolated from manure. They grow on a medium containing 0-3 per cent. NaNO3, sugar, and the necessary salts. Of the nitrogen offered to them, 82-7 per cent, is set free (in some species as much as 99 per cent.), the rest being used up in their cell-substance. Although it is probable that the importance of these denitrifying organisms in agriculture is very small, it cannot be denied that they occur, and they certainly mar to some extent the clearness of our conception of the processes by which nitrogen circulates through organic creation. Just as the nitrifying organisms have their converse in the denitrifying, so have the sulphur bacteria theirs in the desulphurizing species. A spiril- lum (S. de sulfur leans] has been isolated from ditches and sewers, which sets free the SH2 from sulphates. The complete life-history of this organism is not known (75). There can be little doubt that many other biochemical processes are going on in nature besides those we have been considering, and mineralogical chemistry will have to turn its attention to bacteria. Possibly we shall even find prototrophic species which attack silicates. The reader may be left to ponder further on the subject. CHAPTER XII THE CIRCULATION OF CARBON DIOXIDE IN NATURE 1. Introduction ; Organized Ferments and Enzymes ; Races of Ferment Organisms ; Fermentation of Alcohol and of Acids ; Optical Activity and Fermentation. THE ultimate source, from which directly or indirectly all organisms draw their carbon, is the carbonic acid of the atmosphere. In the circula- tion of this gas bacteria play a not less important part than they do in the circulation of nitrogen. Animals, it is well known, cannot assimilate CO0 ; they are dependent for their carbon entirely upon plants. Among these again only the chlorophyll-bearing forms and the red, brown, and blue-green algae are able, with the energy of the absorbed sunlight, to fix the atmospheric CO.,*. From it the plant builds up the organic compounds, with or without nitrogen, that constitute the physical basis of all life upon the earth. Since this assimilating and fixing of carbonic acid is going on without ceasing, there is an evident necessity, if terrestrial life is to endure, for the continual return of carbon in the form of CO2 to the atmosphere, and such restitution does as a matter of fact take place. A certain amount is given back at once by the plants themselves in the process of respiration, and another portion is being continually poured out into the air in the breath of man and animals. All the remaining carbon fixed in the tissues of plants and animals is finally liberated at the death of the organism by the activity of bacteria and other microbes. Those tissue elements which contain nitrogen as well as carbon undergo putrefaction, and the carbon escapes as CO2. The innumerable non-nitrogenous constituents on the other hand — carbohydrates, glucosides, alcohols, organic acids and fats — are * The prototrophic saltpetre bacteria constitute the sole and only exception to this uilc. io8 BACTERIA AND THE CARBON DIOXIDE CYCLE not putresciblc. They are broken up by ' fermentation,' carbonic acid being again the final shape in which the carbon is set free*. In nature, fermentative organisms are more widely distributed even than putrefactive. With these they labour to disintegrate and remove dead animals and plants. But in addition to being indispensable scavengers they are of prime importance to man in numerous industries — in the pre- paration of butter- milk, cheese, ' sauerkraut,' and bread, and in the manufacture of alcohol. Some species, on the other hand, are dangerous enemies to the dairyman and the brewer, and in many technical processes bacteria play a part useful or injurious as the case may be (77). There is a diversity of opinion as to what shall be understood by the word fermentation. Some would apply it to every kind of chemical change arising from the activity of micro-fungi, and would include putrefaction, nitrification, and the oxidation of SH2 by the thiobacteria, in short all biochemical processes. From this it is but a short step to regard human life itself as a fermentation. In the author's opinion, a more restricted application is desirable, both in view of the history of the word and as inducing to clearness. The word ' fermentation' will therefore be used here to designate the decomposition by means of particular living organisms — the fermentation organisms — of non-nitrogenous organic compounds, particularly carboJiydratcs. Given fermentable substances, the necessary conditions for fermentation are a supply of food-stuffs (above all a special source of nitrogen) for the micro-organisms and, as in putrefaction, moisture and a suitable tempera- ture. Even before the nature of fermentation was well understood, we knew that in addition to the fermentable substance a certain something, a ferment, was necessary for the production of the phenomenon, and it was thought by many to be a chemical body. But the classical researches of Pasteur (78) showed that every fermentation was the work of some living organism, a fermcntum I'ii'iim, and not of a chemical ferment or enzyme. We have there- fore to distinguish between living or organized ferments and unorganized chemical ferments or enzymes. The enzymes (79) are substances secreted by cells (e.g. pancreatin, pepsin, ptyalin), and are capable, even in the absence of the cells that produced them, of causing specific chemical changes in certain organic substances. The organized ferments are living cells. They also are able to set up chemical changes in organic bodies, but only so long as they, the cells, are alive. Organized ferments and enzymes have much in common. In each case * In qualification of this somewhat broad statement it should be mentioned that very little is known as yet concerning the biochemical decomposition of fats (76). Preliminary experiments seem to have shown that certain bacteria (cholera, typhoid, B. pyocyaneus) are able to split olive-oil or beef-fat into glycerine and fatty acids, thus making them accessible to fermentation. AND ENZYMES 109 the chemical change produced is specific, is of one kind, and of one kind only. In each case too it is effected apparently without any expenditure of energy, although to produce similar changes in our laboratories we have to resort to very powerful means such as the employment of high tempera- tures, intense chemical reactions, and, in many cases, we cannot reproduce the changes at all. An instance will make these two points clearer. In plants, starch is converted into sugar by the enzyme diastase without apparent effort, whilst in the laboratory we can only cause this change by boiling with mineral acids. But, whereas boiling with acids will effect very many other changes, diastase is powerless to perform any but the one alteration of starch into sugar. We can convert sugar into lactic acid by heating with alkalies ; the lactic bacteria can do the same by their fermentative power, but are unable to produce butyric acid. Some of the biochemical processes of micro-organisms, the conversion of sugar into alcohol for instance, cannot be exactly imitated by chemical means. Finally, neither enzyme nor organic ferment disappears in effecting such changes, as a reagent disappears in a chemical reaction. There is no definite relation between the quantity of ferment used and the quantity of substance fermented, an enzyme like pepsin, or a yeast, being able to ferment a hundred or a thousand times its own weight of fermentable sub- stance. The chief and fundamental difference between enzymes and organized ferments lies of course in the fact that the latter are living beings able to grow and multiply, increasing the more rapidly the more food and ferment- able material are placed at their disposal. This the enzymes cannot do, for they are lifeless chemical compounds, although they have, it is true, many points of resemblance to the proteids. They are extremely unstable com- pounds, and when dissolved in water their fermentative power is destroyed by a short exposure to 50° — 6o°C., just the lethal temperature of most sporeless micro-organisms. But towards protoplasm poisons their behaviour is quite different from that of the organized ferments. Arscnious acid, phenol, salicylic acid, ether, and chloroform in concentrations which paralyze the organized ferments leave the enzymes unaffected. Chloroform however seems to affect some enzymes in a short time. As regards the nature of the chemical change produced by enzymes and organic ferments respectively, there is a broad and fundamental difference between the two. The enzymes exercise solely a Jiydrolytic influence. That is to say, they cause the molecules of insoluble compounds to take up water and to separate into less complex molecules of a different constitution, the resultant substances being soluble in water. Diastase, for instance, con- verts one molecule of starch into one molecule of grape sugar : i io BACTERIA AND THE CARBON DIOXIDE CYCLE In the same way, invertasc, an enzyme secreted by yeast cells, converts one molecule of cane sugar into one molecule of glucose and one of fructose : C12 H22OU 4- H2O = C6H1;, Oc + CCH12O6. The peptic enzyme (pepsin) of the stomach changes insoluble proteids into soluble peptones and albumoses. All tJicse reactions arc complete in themselves, and there are no by-products suck as CO2 or other gases. The action of organized ferments is quite different. By them the highly complex molecules of organic compounds are split up, and numerous substances of a totally different character arise, generally with the evolution of gases and other by-products. For this reason it is impossible to represent the reaction by a simple chemical equation. Ordinary alcoholic fermentation, for in- stance, cannot be written CGH]L,OC = 2C,H00 nor butyric fermentation, for, as we shall see presently, numerous other compounds arc formed. Carbonic acid being evolved in almost all instances, it is customary to designate each process by the name of some other conspicuous product, as the preceding examples show. A by-product of one kind of fermentation may be the chief product of another kind. Acetic acid, for instance, is a by-product of alcoholic fermentation, but the principal substance formed in vinegar fermentation. The organized ferments are often regarded as comprising three classes of micro-organisms, — yeasts, bacteria, and moulds. The last- named, however, are of rare occurrence, e. g. the mucor yeast that causes the decomposition of wine. The so-called ' aspergillus yeast,' used in China and Japan in the preparation of soy and sake, works only by means of an enzyme that it secretes, the alcohol being produced, not by the aspergillus, but by a true yeast. Similarly in ragi, the 'yeast' of the arrack industry, there are present both a true yeast and a mucorine fungus (RJiizopus Oryzae). The function of this latter is to ' invert ' the rice starch and make it accessible to the yeast (80). Mould fungi are sometimes employed on an industrial scale in the preparation of citric acid (81). The discrimination of the various species of fermentative organisms is of very recent date. When Pasteur first showed that fermentation was the work of living cells, we had not the technical means which we now possess of distinguishing closely related species from each other. It was sufficient to assume that every fermentation had its own peculiar fungus, a Bacterium aceticnm in vinegar fermentation, a B. butyricum in butyric fermentation, and so forth. In the same way it was not until the publication of Hansen's A.\D HKKEDS OF BA( Tl'.RIA 1 1 1 revolutionary investigations on yeasts that many different species of SaccJiaro- niyccs were recogni/cd. The SaccJiaromyccs ccrci'isiac of beer- wort, and the vS". cllipsoiiicns of vinous fermentation, and a few others, were the only species known. To-day the species S. ccrci'isinc is divided into hundreds of ' races ' or varieties, and the same has happened to the wine ferments. In these technical processes, some of which arc as old as human culture itself, changes have been going on for thousands of years similar to those changes we have caused artificially and intentionally in our domesticated plants and animals. But while we have no difficulty now in distinguishing from one another the innumerable kinds of cultivated plants, the determination of racial characters among micro-organisms is a task of great difficulty. Morphological data are almost useless, and we have to fall back upon physio- logical peculiarities such as the degree of warmth required (different optima), the nature and amount of by-products, or the specific fermentative power. FlG. 24. Fermentation bacteria, a-c, acetic bacteria trom E. Chr. Hansen. a, Bacillus ace/i ; &, B. Pas- tc nrian its , c, B. KutsinfianUS \ d, B. acidi lactici\ the commonest cause of lactic fermentation ; e, Closiridium biityricum, an anaerobic butyric ferment, ' granulose ' bearing ; to the right spore in spindle-shaped rod. _/| PlfC- tridhun pahidosuni, anaerobic ferment from marsh water, corresponds in form to the methane bacteria, and to some of the butyric bacteria. Magn. a-f 1000. Moreover, it must not be forgotten that new varieties are continually arising, old ones dying out and being replaced by others with recently acquired powers. The shortness of the time necessary for the production of a new race of organisms is shown by such plants as the chrysanthemum and the potato. Although the culture of the potato is only two or three hundred years old, there are no less than five hundred varieties in exis- tence, distinguished by shape and colour, by their flavour, by the relative amount of albumen and starch, and other points. Among bacteria, too, there are forms that, like the yeasts, have been cultivated from time immemorial — in cheese-making, for instance, and in vinegar fermentation. These are now cultivated in pure form in our laboratories. The difficulty of classification among micro-organisms is further increased by the fact that many morphologically different species possess ii2 BACTERIA AND THE CARBON DIOXIDE CYCLE similar zymogenic powers. There are a dozen different species of bacteria that produce butyric acid and lactic acid. And, inasmuch as the descrip- tions given are often far from exact, the student of these species finds himself lost in a pathless forest of forms. For this reason it will be impossible here to describe more than a few species. Some of the fermentation bacteria can acquire pathogenic properties. Two of the butyric ferments found in soil are known to be the cause of disease, Bacillus Chauvoei giving rise to ' quarter-evil ' in cattle, and another form originating malignant oedema. The notorious B. coli communis is a ferment organism in so far that it splits up grape sugar into lactic acid, succinic acid, and ethyl and propyl alcohol, carbonic dioxide arising in the process. By far the greater number of ferment organisms are, however, harmless, and, considering the millions we take inside us every day in milk, cheese, and other food, it is satisfactory to know this. The chemistry of fermentation is clearly understood only in those cases where the process is oxidatory. The acetic fermentation belongs to this category, the alcohol being combined with the oxygen of the air to form aldehyde and water, the aldehyde being further oxidized to acetic acid, and this again, if the process be allowed to go on, burnt up to carbonic acid and water. The changes may be represented by equations thus : CH3CH0HO + O =CH,COH + H.,O CH3COH +0 =CH3COOH CH3COOH +4O = 2CO2+2H,O This fermentation is evidently allied to the respiratory process, and the similar oxidatory processes of the nitrifying and sulphur bacteria. It differs from most fermentative changes in the small number of resultant substances, but is, of course, just as much a phase in the circulation of carbon as any of the more complex processes, where large numbers of by- products arise and obscure our view (see Chap. XIV). Of the fermentations of monacid alcohols only the acetic fermentation of ethyl alcohol is of industrial importance (82). Alcoholic liquids, such as beer or wine, if left exposed to the air in a warm place acquire a strongly acid reaction, and become covered with a white skin or pellicle. This consists of the ' vinegar bacteria,' which slowly oxidize the alcohol to acetic acid, and finally to CO, and water. Sometimes the pellicle consists not of bacteria but of a yeast (Mycoderma aceti), which transforms the alcohol at once into CO2 and water without the intermediate stage of acetic acid. The old form, Bacillus aceti, has been divided by Hansen into three different species, B. aceti, B. Pastenrianus, and B. Kiitzingiamis (Fig. 24). All three are morphologically very similar, non-motile medium-sized rods often growing out into chains, of which the pellicle mainly consists. They are unable to grow in fluids containing more than 14 per cent, of alcohol. ACETIC FERMENTATION 113 Temperature maxima, and some fine differences of shape, distinguish the three species from one another. B. accti is stained entirely yellow by iodine, but in the two other species the gelatinous mass that holds the cells together in the pellicle takes on a blue tint, the cell remaining yellow. Whether a carbohydrate is present is doubtful, and must remain so as long as we are ignorant of the composition of the cell membrane from whose outer layers, by inhibition of water, the jelly is produced. Cellulose is said not to be present. The optimum temperature for acetic fermentation is 34° C., the maxi- mum 42°, the minimum 4°-- 7°. Approach to the maximum gives lise in all three species to involution forms (Fig. 14, c and d, p. 27). The borders of neighbouring cells become indistinct, the cells themselves swell up to pear- or spindle-shaped bodies, and short branches arise on the filaments. Even at the optimal temperature involution forms arise as soon as acetic acid begins to accumulate, and when about 14 per cent, is present all growth stops, and the bacteria die. As a concrete example (83) of the activity of these bacteria it may be mentioned that B. Pastcurianns in 125 c.c. of lager beer (3-7 per cent, by volume of alcohol) formed in seven days, at 34° C., 4-2 grm. of acetic acid. The alcohol had disappeared, and the acetic acid was finally oxidized to CO2 and water. In a parallel culture, after twenty-one days, only 0-7 grm. acid was present. In the manufacture of acetic acid the process must, of course, be stopped at the proper time in order to prevent loss of acid by further oxidation. In vinegar factories the so-called vinegar plant consists of a zoogloca mass of acetic bacteria, of which there are doubtless many races. The usual methods of vinegar-making all aim at bringing the alcoholic fluid as much as possible into contact with the air, either by running it into large vats or by letting it flow over wood-shavings in barrels. These methods originated at a time when the process was supposed to be a purely chemical oxidation effected by the air. But the bacteria lying upon the surface of the shavings are the cause of the change ; the free access of air promoting it only because the vitality of the bacteria is increased thereby. Any alcoholic fluids, such as cider, dilute brandy, or wine, can be used to make vinegar, but the taste varies according to the nature of those con- stituents of the liquids that remain unaltered by the process. As a by-product, acetic acid arises in many other fermentations, the vinous, the lactic, and the butyric, for example. In fluids containing no alcohol, however nutritious these may be, the acetic bacteria cannot thrive. They can make use of ammonia salts as a source of nitrogen, but obtain this element in the liquids used for vinegar- making from proteid substances. Whether the alcohol is used for respira- tion only, or whether its carbon is in part seized upon by the bacteria as food, is not known. rlS<-»EK I ii4 BACTERIA AND THE CARBON DIOXIDE CYCLE Other fermentations of monacid alcohols have not been described, but there can be little doubt that such occur. Fermentation of higher alcohols (84) has been observed in the case of a motile bacterium isolated from sheep's dung, Bacillus cthaceticns. A culture of this micro-organism in 60 grm. glycerine formed 7-52 grammes Ethyl Alcohol, 3-88 „ Acetic Acid, 0-06 „ Succinic Acid, Carbonic Dioxide, Free Hydrogen, Traces of Formic Acid. 24-19 grm. of glycerine was unchanged. The same bacterium causes similar changes in mannite, but not in its isomer dulcite. The so-called capsule bacillus Pncujnococcus of Friedlander ferments mannite also (with the same products), but not dulcite. Bacillus etJiacctosnccinicus (also isolated from dung) ferments both mannite and dulcite. In cultures eighty-five days old that contained, besides the necessary nutriment, eight grams of fermentable substance, there was formed From Dulcite. From Mannite. Ethyl alcohol I -oil 1-03 Formic acid . . . . . 0-128 0-263 Acetic acid 0-322 0-308 Succinic acid 0-264 o>29 CO2 1-05 1-1 Free H 0-04 0-03 Unfermented residue .... 2-62 3-2 These figures are of some interest, showing that the chief products are alcohol and carbonic dioxide, and proving that the formation of ethyl alcohol is not a monopoly of the Saccharomyces *. Glycerine also is capable of fermentation in other ways, butyl alcohol and butyric acid arising among the products (B. orthobutylicus, Chap. XIII). As examples of the fermentation of fatty and aromatic acids, given in the form of neutral salts, it may be mentioned that acetic acid is oxidized, by the very organisms that produce it, to CO2 and H2O, and that the dextro-rotatory tartaric acid of wine is converted by various bacteria into formic, acetic, butyric, propionic, lactic, and succinic acids. It is to the activity of such bacteria that the diminution of acidity of wine is due, and probably some of the diseases of wine also. The malic acid in cider is also oxidized by bacteria to acetic, propionic, and butyric acids, carbonic dioxide, and water; and similar processes are known in the case of citric, h. Fit-iamts from hay-infusions also produces ethyl alcohol. SEPARATION OJ- ori'lCAL ISOMERS 115 succinic, and other acids. Lactic acid, itself a product of many carbo- hydrate fermentations, is converted by butyric bacteria into butyric acid, carbon dioxide, and free hydrogen. The biochemistry of all these processes is as yet but little understood (Chap. XIII). Very remarkable decompositions of the optically 'inactive' varieties of organic acids are effected by some bacteria*. These consist, as is known, of equal parts of the dextro-rotatory and laevo-rotatory acids, which twist the plane of polarized light to right and left respectively. In a solution of the ammonium salt of racemic acid, for instance, only the dextro-rotatory variety is used by the bacteria, the laevo-rotatory modi- fication being thereby as it were set free. The inactive varieties of lactic and mandelic acids are biochemically separable into their components in the same way (85). The profound chemical disintegration that these changes seem to show is only apparent. As a matter of fact they are only selective changes, only one of the two active compounds of the salt being used up. One of the mucigenous bacteria, for instance, uses up fumaric acid, but leaves the stereo-isomeric compound, malei'c acid, untouched. A similar phenomenon, which may be termed the converse of those just described, is exhibited by a variety of B. coli communis wrhich ferments grape sugar into lactic acids differing in rotatory power according to the source of nitrogen employed. If it be supplied writh phosphate of ammonia, laevo-rotatory acid is formed, but if the nitrogen be given to it in the shape of peptone the dextro- rotatory variety appears ; whereas the lactic acid produced by other fermentations is generally inactive. We must for the present be content to record these facts — an explana- tion is not possible. The stereochemical hypotheses, with which modern chemistry seeks to unravel the molecular structure of these isomeric com- pounds, throw no light whatever at present on the biochemical phenomena associated with them. Some of the mould-fungi act in a similar way. I 2 CHAPTER XIII THE CIRCULATION OF CARBON DIOXIDE IN NATURE (continued) 2. Bacterial Fermentation of Carbohydrates. THE bacteria of lactic fermentation are very widely distributed in nature, and play an important role, not only in dairy work, but also in numerous other industrial processes — often as indispensable auxiliaries, but sometimes as dangerous enemies and intruders. Grape sugar, cane sugar, and milk sugar are all accessible to lactic fermentation. Other sugars (e. g. maltose) are not, nor are carbohydrates like starch or cellulose. All these must be changed into a fermentable form before they can be broken up by the lactic acid bacteria, which themselves produce no such enzymes. The lactic fermentation is an aerobic process whose optimum lies between 30° and 35° C. (for some species between 47° and 52° C.). It can go on for a long period only when basic substances, such as calcic carbonate, are present to fix and neutralize the lactic acid as it arises, a very small amount (0-15 per cent.) of free acid arresting the growth of the bacteria. Eighty per cent, of the fermentable sugar is converted under favourable circumstances into the so-called ' fermentation lactic acid,' optically inactive ethylidene lactic acid, besides which are formed varying amounts of acetic acid, optically active lactic acid, carbon dioxide, and other by-products. The power of forming lactic acid from sugar is common to many bacteria (e. g. the cholera and most other vibrios, B. prodigiosns, various species from the intestines of infants, and many brewery Sarcinae). But by lactic acid bacteria are generally understood those forms which are the regular cause of the acidification of milk in dairies. These micro-organisms were formerly looked upon as belonging to one species, Bacterium acidi lactici (86), but more recent investigations have shown that not one species but many are at work, sometimes one kind, sometimes another, prevailing. BACTERIA IN MILK 117 A very common form is a short non-motile rod (i-2/x by 0-5 jx), facultatively anaerobic (spores unknown), and not liquefying gelatine (Fig. 24, d}. It is known by various names, Bacillus aerogcncs, JB. acidi lactici, &c., and comprises probably several races of one species. They may be called the typical bacteria of lactic fermentation. Mingled with them are found, sometimes in great numbers, the spherical or ovoidal cells which cause the coagulation of milk. The lactic fermentation of mash in distilleries is caused by yet another form, B. acidificans longissimus (87). This bacterium is about i ju broad and something more than 2-5 //, long. The number of lactic acid bacteria is great and their determination difficult. The manifold importance of the lactic ferments can be best illustrated by some concrete examples. J\Iilk and other Dairy Products (86), etc. Cow's milk, being neutral or slightly alkaline, and containing as it does from four to five per cent, of milk sugar, 4 per cent, casein, and 0-7 per cent, of the necessary mineral salts, is an excellent nutritive medium for bacteria. They multiply in it very rapidly, and within a few hours after it is drawn are present in enormous numbers (from 100 to 6,000,000 per c.c.). As might be expected, the numbers vary greatly according to the cleanliness of handling during milking and afterwards. In consequence of the favourable conditions thus offered for the multiplication of bacteria, the sterilization of milk, particularly for children, has become a matter of great importance *, and numerous inventions have been put forward to attain a thorough and efficient sterilization. But milk, although sterile when drawn from the udder, becomes infected with micro-organisms at once on contact with the outside world, and invariably contains spore- bearing bacteria. Some of the spores are so resistant that even boiling for an hour, or an hour and a half in a Soxleth boiler, does not destroy them, so that at present it seems impossible to render milk absolutely sterile without exposing it to heat so great that its chemical characters are altered. Fortunately, the majority of the bacteria in milk are sporeless cells which are killed by boiling for five or ten minutes, and for this reason expensive sterilizing apparatus is being largely discarded in favour of the old domestic method of boiling the milk, and then keeping it as cool as possible, so that the uninjured spores may not germinate (88). Besides the predominating lactic bacteria, there are always present species that secrete an enzyme resembling rennet, and frequently chromo- 2cnic forms in small number. o Since the presence of pathogenic germs in milk may become a source * In Germany a matter of State and municipal control. ii8 BACTERIA AND THE CARBON DIOXIDE CYCLE of danger (89), experiments have been made to determine their behaviour in milk. The bacteria of typhoid, anthrax, glanders, tubercle, diphtheria and cholera grow well, without causing any change in the appearance of the milk that would suggest contamination — ordinary market milk does not look as though it contained millions of bacteria — and not until some time has elapsed does the milk begin to coagulate in consequence of the acid produced. The anthrax bacillus produces acetic and caproic acids. Whether the milk of diseased cows contains the specific bacteria of the infection is not certain in all cases. In tuberculosis, however, tubercle bacilli have been repeatedly found in the milk. The acidification of milk for the preparation of cheese made from the ' acid curd ' is the work of the lactic bacteria already mentioned. The acid they produce precipitates the casein — the milk ' coagulates.' In the preparation of ' rennet curd ' the same end is attained without acid by the use of rennet, an enzyme prepared from the stomach of calves. In both cases, the casein freed from the serum of the milk (whey) constitutes the ' curds ' from which cheese is prepared. Numerous ' diseases ' of milk are caused by bacterial action. Not infrequently it coagulates without turning acid ; this is the work of species (Tyrothrix, found in cheese) which secrete an enzyme resembling rennet. Pigment bacteria often make their appearance spontaneously and cause the milk to have an unnatural colour. Red is produced by B. pro- digiosits and some Sarcinae, blue by the harmless B. cyanogcnns^ a small motile rod that grows on agar in dark-blue or bluish-grey crusts according to the food-stuffs available. From yellow milk also several pigment bacteria have been isolated. These coloured milks have generally become more or less acid. ' Ropy ' milk is the work of certain mucigenous bacteria that will be described later on. Bitterness in milk is produced especially by peptone-secreting bacteria that possess very resistant spores. Butter is always rich in bacteria. A specimen of Munich butter contained from six to twenty-five million per gram. Since the butter contains from 0-5 to 1-5 per cent, lactose and other food-stuffs, changes may be set up by the formation of lactic and butyric acids. The butter is thereby rendered rancid, but it must be remarked that the rancidity of butter is in most cases the result of the oxidation of the butter fat to fatty acids (butyric and lactic) by the free oxygen of the air accelerated by exposure to light. The peculiar aroma, too, that renders some butters so tasty, is the product of certain bacteria that are now cultivated in dairy laboratories and added to the fresh butter (90). A process of great complication, the details of which are difficult to follow, is the ripening of cheese. This is the work of many kinds of bacteria, which are therefore present in cheese in enormous numbers (91). In a gram of German cheese from five to six million germs have been BACTERIA AND CHEESE 119 found, in the same quantity of Swiss cheese one million, in other sorts still more. In the preparation of Roquefort cheese a mould fungus (Peni- cilliuni glancnni) is introduced with mouldy bread, and forms the well- known green patches in the cheese. In other cases, forms like Oidium and also yeast fungi play a part. Not all the bacteria of cheese are equally active in the ripening process ; many are only useless intruders, others perhaps contribute to the finer shades of flavour. The main part of the process is the work of the lactic and butyric ferments. Forms that were once supposed to be specific 'cheese bacteria' (TyrotJirix, Duclaux, allied to B. snbtilis] do not play the important part that was attributed to them. How difficult it is to determine the share which the various species have in the process, is shown by the fact that in some cheeses no .less than nineteen different bacteria and three yeasts have been found. In other kinds the flora is still more varied, and it changes, moreover, with the different stages of the ripening process. This is the reason that, in spite of numerous and careful investigations, the bio-chemistry of cheese has but few results to show, and those contradictory. Even the qualitative composition of cheese varies, and a quantitative analysis is as yet no more possible than in the case of putrefactive processes. Emmenthaler cheese contains lactic, butyric, and phenylamidopropionic acids, leucin, tyrosin, ammonia, casein (partly unchanged, partly in the form of soluble albumoses), fats, and fatty acids. The fresh curds from which cheese is prepared contain chiefly three substances, whose gradual alteration constitutes the ripening: i. Carbo- hydrates (milk sugar) ; 2. Proteids (casein and paracasein) ; 3. Fat. The milk sugar is converted first by the lactic bacteria, and afterwards by the butyric bacteria, into organic acids, CO2, and free hydrogen. It is these gases which cause the cavities in the cheese. The casein is changed, by the action of the enzyme secreted by the bacteria, into an albumose-like body (improperly called caseoglutin), and a small portion of it further split up into tyrosin, leucin, ammonia, and phenylamidopropionic acid ; but genuine putrefactive products, like skatol or indol, are not formed. The ripening of cheese cannot therefore be regarded as a true putrefaction, but only as an allied process in which fatty acids arise. The conversion of the casein goes on very gradually until, by the time the ripening is finished, there is none left in an unchanged state. It is not known precisely what species of bacteria are instrumental in effecting this alteration. Fat is not formed from casein, and the butter remaining in the curds is for a long time unaffected, but towards the end of the process it is split up into glycerine and fatty acids. Of these phenomena the two of chief importance are the fermentation of the milk sugar and the alteration of the casein. Another derivative of milk, kephir, is produced by the united action of lactic acid bacteria and a yeast (SaccJiarcuiyccs). Kephir is a weakly 120 BACTERIA AND THE CARBON DIOXIDE CYCLE alcoholic effervescent beverage that has been used for centuries in the Caucasus, where it is usually made from mare's milk. With us cow's milk- is generally employed. The ' kephir grains ' with which it is prepared con- tain both the bacterium and the yeast. The yeast cells secrete a peculiar enzyme (lactase) that converts the milk sugar into grape sugar, which is then fermented to alcohol and CO2. The lactic bacteria, besides giving the pleasant acid taste to the drink, cause the precipitation of the casein in a very finely flocculent, easily digestible form. Acetic and succinic acids arise as by-products (91). In distillery operations (92) the development of butyric bacteria was formerly much dreaded. In the preparation of sweet mash from green malt the heating for two hours at 70° is naturally insufficient to kill the spores. Experience showed, however, that a certain degree of acidity in the mash prevented the development of butyric bacteria without injuring the yeast cells, and careful investigation showed that the acidity was caused by the lactic ferment. In practice the end is attained by inoculating some of the sweet mash containing the yeast to be used with pure cultures of a lactic bacterium, and keeping it at 50° C. (the optimum for these organisms) for some time before adding it to the main body of the mash. At 50° C. the lactic bacteria flourish and produce as much as i per cent, of lactic acid, and the growth of the butyric organisms, whose optimum is 40° C., is inhibited both by this temperature and by the lactic acid. This important application of lactic fermentation is now largely super- seded by Effront's hydrofluoric acid method. The yeasts are far less sen- sitive to acids generally than the bacteria, and by continued culture with increasing quantities of acid they may be accustomed to such large amounts that the bacteria are reduced to a minimum, or even quite suppressed. In a few months the alcohol yeasts may be accustomed to as much as thirty milligrams of hydrofluoric acid per hectolitre mash, ten mg. sufficing to kill the bacteria. Other poisons also have been tried ; formaldehyde, for instance, which seems more efficacious even than hydrofluoric acid. For electrical sterilization see p. 72. The spoiling and ' turning ' of beverages and articles of food through lactic fermentation is a common occurrence. Beer must contain more than 7 per cent, alcohol if it is to be safe from the attacks of the lactic bacteria, which, if they once get a foothold, soon cause it to ' turn,' making it turbid and giving it a bad taste. Wine, too, is often spoiled by the same organisms, and may contain as much as 2 per cent, lactic acid, derived from the fructose. The acidity of wine is, however, more often caused by the acetic bacteria than by the lactic. Boiled vegetables are not infrequently soured by both lactic and butyric fermentation. The various methods of fodder preparation (93), brown hay, sour fodder, and sweet ensilage, are fundamentally lactic fermentations by which the hay nUTYlUC ACID BACTERIA 121 is made both durable and more tasty for the cattle. Sauerkraut is another foodstuff that is the result of such fermentation. In all these cases butyric fermentation occurs at the same time. Butyric Fermentation (94). The butyric fermentation, a strictly anaerobic process, is no less widely distributed in nature than the lactic. Its importance for the general theory of fermentation will be considered in the next chapter. There are several means of obtaining rough, impure fermentations of butyric bacteria from which, by growing in hydrogen or by other anaerobic methods, pure cultures may be derived. It suffices to place some peas in a saccharine nutritive fluid and close the culture flask with a cork through which passes a tube whose outer end dips under water. In a few days at 30° or 40° active fermentation sets in, large quantities of gas are given off, and an odour of butyric acid is apparent. Another method is to boil a mixture of 5 grm. grape sugar and 5 grm. powdered fibrin in 100 c.c. water, and in- oculate the liquid, whilst boiling, with a little garden earth. After forty-eight hours at 35° the fermentative process is going on rapidly, and is caused almost entirely by one species of bacterium, the Granulobacter saccliaro- bntyricus of Beyerinck. Just as was the case with the lactic fermentation, the butyric fermenta- tion was supposed formerly to be the work of one species of micro-organism only, the Vibrion butyriq?teof*Pa.st&ur(Ainylobacter bntyricus, van Tieghem). There can be no doubt, however, that these terms have both morpho- logically and physiologically only the value of collective designations, as is the case with the Clostridium butyricum of Prazmowski. Some twenty different forms of butyric bacteria have been more or less precisely de- scribed, and the number could be probably reduced to two or three. Many are distinguished by the granulose reaction (Beyerinck's biological genus Granulobacter), and some are characterized by the alteration in shape of the cell during sporulation, a process which sets in with great regularity towards the end of fermentation (Fig. 24, e, /). The commonest form of the spore-bearing cell is the spindle shape (Clostridium), but some species swell at the end (drum-stick, Plectridium). Almost all arc comparatively large cells (0-5-1/1 by 3-ioju) actively motile and pcritrichous (Fig. 24, c,f}. Granulobactcr saccJiarobutyricns and G. lactobutyricus (anaerobic Clos- tridia with granulose), and Bacillus orthobutylicus (an anaerobic Clostridium with no granulose), all produce considerable quantities of butyric acid, with some CO2, free hydrogen, acetic, and traces of other fatty acids. B. ortJio- bntylicns ferments carbohydrates and other compounds of many different kinds ; glycerine, mannitc, glucose, cuvert -sugar, cane-sugar, maltose, lactose, arabinose, starch, dextrine, inuline (not trehalose), erythrite, and gum arabic. 122 BACTERIA AND THE CARBON DIOXIDE CYCLE In some cases, of course, the fermentation must be preceded by inversion by enzymes. The other two species mentioned are, as their names imply, more particular, and need grape, cane, or milk sugar as food. 2-4 grams of glucose fermented by B. orthobutylicns yielded in twenty days Grams. 0-842 Normal Butyric acid 0-264 Butylic alcohol 0-229 Acetic acid besides free hydrogen and C(X. These gases increased by continued fer- mentation, probably showing that the organism can split up its own pro- ducts into still simpler compounds down to CO2. A granulose-bearing species from the soil, described by Beyerinck as Granulobacter butyricus, is particularly worth notice, since it forms from maltose, not butyric acid, but butylic alcohol with some CO2 and free H. Not infrequently butyric acid arises as a product in putrefactive pro- cesses, and it seems that some butyric bacteria (B. butyricns, Hueppe) have true saprogenic properties, and can break up proteids. Other species, like B. orthobutylicus, can form butyric acid from peptone only in the presence of one of the above-mentioned non-nitrogenous bodies. Milk soured by B. lactis is often infected by butyric organisms which disintegrate both the remaining milk sugar and the lactic acid that has arisen from it. The fermentation of the calcium salt of lactic acid ex- hibits this change in an uncomplicated form *. Cellulose Fermentation. In manure heaps and in the rotting vegetation at the bottom of lakes and ditches there is going on side by side with the disintegration of carbohydrates in plant tissues another process, a special fermentation of cellulose known as methane or marsh gas fermentation (95). The organ- isms which effect this change first of all ' invert' the cellulose (i. e. break it up into a sugar) by means of an enzyme and then ferment it to methane CH4 and carbon dioxide, fatty acids arising as by-products. Methane, mixed with free hydrogen and CO2, rises to the surface when the mud at the bottom of ponds is stirred up with a stick. There seem to be a considerable number of methane bacteria. The Vibrio rugula (anaerobic, granulose-bearing) probably is one. Another species (a delicate anaerobic motile Plectridiuni) that has been isolated from * For butyric bacteria in cheese-making see p. 119; their distribution in nature, p. 135 ; their assimilation of nitrogen in the soil, p. 96. TECHNICAL FERMENTATION 123 sewer contents is able to ferment filter paper (pure cellulose). The paper becomes soft and transparent, and is finally completely dissolved. Methane bacteria are at work also in the intestines of herbivores and of man, and cause distcntion by the gases they produce. Mucilaginous Fermentation (96). Wine, beer, and milk sometimes become ' ropy,' slimy or ' stringy ' ; boiled vegetables, too, sometimes undergo the same change. This is due to the growth of bacteria, which ferment the carbohydrate constituents of the liquid, and whose chief product is mucilage, carbon dioxide, free hydrogen, and the inevitable fatty acids arising as by-products. In some cases the nascent hydrogen combines with dextrose to form mannite. The mucilage is a gum-like carbohydrate allied to vegetable gums, and having the same composition as cellulose (C6 H10 O5)n. It is not a fermentation product, however, in the true sense ; that is to say, it is not a direct result of the protoplasmic metabolism, but arises through the imbibition of water by the outer layers of the cell-membrane. It is, in fact, similar to the jelly that holds together the cells in a zoogloea. A useful task would be the investi- gation of the nature of the unaltered inner layers of the cell-wall, which consist perhaps of a cellulose-like carbohydrate. A number of mucilaginous bacteria have been described, and the species are said to be different for different kinds of sugars. B, viscosus sacchari is said to flourish only in fluids containing cane sugar, another only in grape sugar (wine), and a third (B. viscosus lactici) in milk sugar. Fermentations in technical processes. Wherever fermentable material is handled on a large scale there is danger of the invasion of ferment bacteria. And in many processes that ' go on of themselves ' we must assume that bacteria are at work. Places where such changes occur, and where the bacteriologist must seek for them, will occur to every one. Some cases, such as the souring of bark liquor in tannery, have already been tentatively examined. It will be worth while to mention a few others. The ''Retting' of Vegetable Fibres (97), such as flax and hemp, is also the work of bacteria. The tissues are allowed to soak for a long time in water, and fermentation sets in, the fibres being freed from the enclosing cells by the solution of the middle layer of the cell-walls. This inter-cellular cement- substance consists of so-called pcctine compounds (pcctinc salts of calcium) related to carbohydrates, which are dissolved by the bacteria. The fibres can then be separated by mechanical means. Up to the present we know only one of the micro-organisms concerned, an anaerobic Flcctridiuin i^n by o-8//), which can use ammonia as a source of nitrogen and 124 BACTERIA AND THE CARBON DIOXIDE CYCLE ferments pectine substances prepared from linseed, pears, or turnips. Cellulose and gum arabic are not attacked, but the power of breaking up other carbohydrates is greatly increased if peptone be supplied instead of ammonia. As to the products of pectine fermentation nothing is known, but they are probably, as in other cases, CO2 and fatty acids. The old idea that the process was one of cellulose fermentation is certainly wrong. In the manufacture of indigo bacteria are considered to play an im- portant part (98). The indigo plant (Indigofera tinctoria] contains a gluco- side Indican which by anaerobic fermentation at 25°-35° C. is converted in from eight to fifteen hours into a sugar (Indigoglucin) and indigo-white. The fermentation is carried on in large vats, the liquid in which becomes blue on the surface where it is exposed to the air. By agitation the whole mass is brought into contact with air, and becomes blue throughout. The micro- organism which ferments the indican is a short capsulated bacillus, $. indigo- genus. If the fluid be sterilized and the bacteria killed, no pigment is formed. The process has not yet been followed in detail. The preparation of tobacco involves, to a considerable extent, the work of bacteria (99). The dried leaves of the plant are damped and laid in great heaps to ' ferment.' By this process various carbohydrates, nicotine, and vegetable acids are converted into CO2, butyric acid, succinic acid, and certain ' aromatic bodies,' whose nature is not precisely known. The proteids are said not to be attacked. Various species of bacteria have been isolated from the tobacco leaves. The ' Havannah ' bacteria are said to differ from those of the German weed, and the inoculation of the latter with Havannah bacteria has been to some extent successful. The process is comparable to the improvement of wines by the inoculation of the grape juice with specific pure yeasts. It is, however, doubtful whether the real aroma of Havannah tobacco can be transferred in this way, since the aromatic compounds of the plant itself have to be considered, not merely those produced zymogenically. In the beet juice of sugar factories, and also in refineries (100), the ' frog-spawn bacterium ' (Leuconostoc mcsenteroidcs) is sometimes a great pest (Fig. 7, b— d}. It is a mucigenous bacterium, and the chemical changes it sets up have been designated as dextrane fermentation, because the mucilage that appears in such profusion is supposed to be similar to a carbohydrate of the beetroot, dextrane. The matter requires further investigation, however. At the optimum temperature (3o°-35°) the micro-organism grows with extraordinary rapidity, and whole vats may be filled with it in the course of a single night *. Besides mucilage, the bacteria produce lactic acid and CO2. The molecules of the fermentable substances are not so completely * In one case a vat of 49 hectolitres molasses, containing 10 per cent, sugar, was filled in twenty- four hours with the spawn-like masses. FERMENTATION OF BREAD 125 disintegrated as in many fermentations. The bacterium is a coccus with fixed planes of division, and, like the alga Nostoc, forms unbranched rosary- like chains embedded in jelly. Cane and grape sugar are necessary for the production of the mucilage. In media containing neither of these carbohydrates, the organism grows as a simple Streptococcus, without any capsule. In bread-making (101), too, we cannot dispense with the help of micro- organisms, which are important agents in the conversion of the nutritious wheat meal into a tasty bread. Yeast, as used by bakers, consists of a mixture of Sacckaromyces cells (the alcohol ferment), and of different species of bacteria. The bacteria, to some extent, prepare the starch for the yeast by enzymes, and contribute to the taste of the bread by the organic acids (lactic and acetic) they produce. The fermentation set up by the yeast gives rise, per kilogram of bread, to about 2-5 grm. alcohol and 2*7 grm. CO2- This gas it is which causes the bread to ' rise,' and the inflation is of course increased by the expulsion of alcohol vapour and steam, which takes place in baking. Some of the fermentation products finally remain in the bread and affect its taste. CHAPTER XIV THE CIRCULATION OF CARBON DIOXIDE IN NATURE (continued) 3. The Yeast Fungi and Alcoholic Fermentation. Theory of Fermentation and Anaerobiosis. Concluding Remarks on the Circulation of Nitrogen and Carbon in Nature. THE most important of all fermentative processes, from an industrial point of view, the alcoholic or vinous fermentation, is the work not of bacteria, but of yeast fungi. Some few bacteria are, as we have seen, able to produce ethyl alcohol, e. g. B. ethaccticns, but in all the great technical fermentations, in the manufacture of beer, wine, and spirits, the ferment organisms are yeasts (Saccharomycetes>Blastomycetes) (102, 103). The yeasts are unicellular non-motile organisms, not cylindrical or spherical in shape like the bacteria, but spheroidal (Fig. 25). The shape of the cells, to some extent of value as a classificatory character, appears at first sight more irregular than it really is. This is due to the remarkable manner of pro- pagation. The yeast cells multiply not by fission into two equal halves, as do bacteria and the cells of higher plants, but by ' budding.' The cell-wall becomes evaginated at one or more points, and the bud-like projections thus formed are shut off from the mother-cell by a new cell-wall. As the young cells do not at once break away, and even begin to bud themselves before connexion with the parent cells is severed, irregular colonial growths arise. The contrast between this mode of reproduction and the multiplication by fission of the bacteria is very great. Among the bacteria each new generation represents half of the parent generation, which in the very act of multiplication ceases to exist as an individual. In the yeasts a small part only is separated from the parent, which continues to live and bud off further descendants. The process of budding, like that of fission, is very rapid ; a new generation may arise in a couple of hours, so that reproduction is little less prolific in the yeasts than in the bacteria. As with bacteria, the successive generations of yeast cells often remain SACCHAROMYCES 127 in close connexion. Hut, inasmuch as the budding is irregular, new individuals sprouting out from any part of the surface of the cell, the colonies or growth forms that arise are also irregular, branching out not only in one plane, but in all directions, without any definite sequence of growth (Fig. 25). The cells are often elongated, sausage-shaped, and the tangled colonies or 'growth forms' have then almost the appearance of CD rp ro 00 \J i. FIG. 25. Saccharomyces. a, Saccharomyces cerevisiae, No. I. 5, Sacch. Pasteuriatitis, No. 3. f, Sacch. ellips- oHeus (wine-yeast), No. i. rf, Sacch. ellipsoideus, No. 2. ^ and f, pellicle growth (pseudomycelmmj of Sacch. tuffs., No. i ; e. at »o -20 or b -7 ; _/; at 15 -30 . g-k, spore-liearing cells; ,f, Sacch. cererisiae, \ ; A, Sacch. Pasteur., i ; j' and k, Sacch e//t/>s., I and 2. /, germination of two free spores ot Sacch. Litdwigii at 18 -20°, from It-tt to right, after 18, 2i>, 26, 28, 29, 30}, and 33 hours respectively. All cultures in beer-wort, luagn. 1000 (trom E. Chr. Hansen). mycelia (Fig. 25, e and g). Such pseudo-mycclial masses arise both immersed in liquids and on the surface. In the latter position they often unite to form membranes or pellicles ('veils'), as we saw was the case with some bacteria. Their mode of origin shows them to be, however, not true mycelia, but only 'growth-forms.' The yeast cells, like those of other plants, consist of a membrane 128 BACTERIA AND THE CARBON DIOXIDE CYCLE enclosing the protoplasmic cell body, which apparently contains a nucleus *. Although very minute, the yeast cells are larger than most bacteria, having on an average a diameter of from 8 /u. to 10 /u. All the yeasts that are of industrial importance are colourless, and their cultures appear white or of a yellowish tinge. There is, however, a pink yeast (Saccliaromyces glu tints, weakly fermentative) that frequently crops up as an intruder on culture plates, and a less common black species is known. Most yeasts under favourable circumstances (free access of air ; surface culture, not immersed ; temperature about 25° C.) form spores. Instead of each cell giving rise to one endospore, as in bacteria, several appear, the protoplasm of the parent-cell dividing into several (generally two to four) rounded masses, each of which becomes surrounded by a new membrane (Fig. 25, g, k). The spores are considerably less resistant than those of bacteria, being killed by a temperature of 62°-7o° C. in five minutes. They are capable of germination as soon as they are formed, but will withstand drying for long periods. The germinating spore begins to bud as soon as the spore-wall is thrown off (Fig. 25, /). Hansen's investigations have shown that in the hands of skilled observers who are familiar with the sources of error, the peculiarities of sporulation and its relation to certain temperatures, are of great importance for the differentiation and determination of species and races. The following table gives the times of sporulation for a ' high ' yeast (S. cerevisiae], for a race of wine yeasts (S. ellipsoidens], and for two races of 'wild' yeasts from the air of a brewery (S. Pastcuriawts). Important points are the optimum and the maximum temperatures, and the time occupied in forming the spores. Temperature. S. cerevisiae. S. Past euri anus. S. ellipsoideus. a. b. 37-5° no spores 36°-37° 29 hours (Max.) 35° 25 hours 3i-5° no spores 36 hours (Max.) 30° 20 hours (Opt.) 30 hours (Max.) 27-5° 24 hours (Opt.) 34 hours (Max.) 25° 23 hours 25 hours (Opt.) 21 hours (Opt.) 18° 50 hours 35 hours 36 hours 33 hours n°-i2° 10 days (Min.) 77 hours 7° no spores 7 days 7 days IT days (Min.) 3°-4° no spores 14 days (Min.) 17 days (Min.) no spores * See Wager, ' The Nucleus of the Yeast-Plant,' Annals of Botany, Vol. XII, Dec. 1898, p. 499. RACES OF YEASTS 129 The cardinal temperatures for sporulation are given and show the value of such physiological data. For the differentiation of very closely related races more subtle distinctions are utilized, such as the shape of the cells, mode of budding, power of fermentation, and particularly the ability to break up various sugars of closely allied chemical structure. Some of the natural species of yeasts are destitute of spores. Hansen (104), guided by the attempt to cultivate a sporeless breed of anthrax bacilli, endeavoured to induce the asporogenous condition among spore-bearing yeasts by similar means, i. e. by cultivation at high temperature. His experiments were successful ; sporulation was suppressed and did not appear in subsequent generations. At the same time the fermenting powers of the yeast were slightly altered. It seemed at first that the attempt to obtain sporeless races had been more successful among the yeasts than with the bacteria, but subsequent observations showed that the phenomena were the same in both cases. When mixed with earth, the sporeless varieties of yeast died out in one year, whereas the unaltered spore-bearing form of the same species lived for three years under the same conditions. This fact alone shows that a general weakening of the organism had set in, and other signs of degeneration were not wanting. But although the attempt to produce races of yeast with new morphological characters has not been an unimpeachable success it is undoubtedly possible to obtain races with more or less permanently altered physiological functions — races that produce more (or less) alcohol than the parent form, or in which the by-products of fermentation are different or present indifferent proportions. In the brewing industries (105) hundreds of such races have already arisen, and give to each particular kind of beer its specific flavour, while Hansen's investigations have led to the application to yeasts of the methods of pure culture, so that the most desirable varieties can be artificially propagated. The wine yeasts also include innumerable races. Almost every kind of wine has its own species of SaccJiaromyces. The secondary products of fermentation are of great importance, particularly the aromatic sub- stances (ethers) which give the ' bouquet.' But, whilst these zymogenic products are undoubtedly influential in determining the flavour of the wine, it must not be forgotten that the aromatic constituents of the grape itself are of still more importance. The pure culture of yeasts of known fermentative properties is a great step forwards, but it is not everything. It will never be possible, for instance, to make a sour inferior Rhine wine into an expensive Johannisberger merely by the use of the Johannisberg yeast (105). In the old methods of wine-making the fermentation was left to ' go on of itself ' ; in other words, it was left to the yeasts accidentally present in the grape juice. The grapes hanging on the vines always have FISCHER K. 130 BACTERIA AND THE CARBON DIOXIDE CYCLE on their skins numerous yeast cells, which multiply rapidly on burst or broken grapes and are carried about by insects from one plant to another. After the grapes have been gathered, millions of yeast cells remain in the soil, where they pass the winter until next year's crop supplies them with fresh food. When the fermentation is to be made with a pure yeast, it is not necessary to kill the Saccharomyces already present in the grape juice by heat ; it is sufficient to add a large quantity of the pure culture, the organisms in which usually easily overcome the less numerous ' wild ' yeasts. The term ' species ' has the same meaning and value among the Saccharomyces as among the bacteria, but it must not be forgotten that the yeast fungi are among the oldest of cultivated plants, and that in the course of ages innumerable varieties and races have arisen. The various yeasts of breweries and distilleries must all be looked upon as varieties of the one species *S. cerevisiae, and a few others, those used in wine- making, as races of 5. cllipsoidens (Fig. 25, a, c, d), a somewhat smaller, thinner form than 5". cerevisiae. The peculiar mode of propagation of the Saccharomycetes characterizes them as a definite and independent group of organisms, whose systematic value is the same as that of any other order of fungi. This independence would never have been challenged had not a similar budding-off of cells been observed in some other fungi (106). Spores of the smut- fungus (Ustilago) if sown in a decoction of horse-dung germinate and protrude a small few-celled pro-mycelium, which then buds off laterally rounded cells just in the same way as yeasts do. The free-lying rounded cells multiply still further by budding, and give rise to cell groups and colonies indistinguishable, as far as appearance goes, from true yeasts. They are, hozvever, quite unable to set up alcoholic fermentation. Again, the filamentous hyphae of some of the mucorine fungi, if grown submerged in saccharine solutions, produce cells which sprout and multiply like yeasts, and even cause a small quantity of alcohol to be formed (Mucor racemosus, M. ercctus, M. circinelloides}. Finally, there are ascomycetes (Exoascns] which give rise to budding cells, and whose mode of sporulation (ascospores) is suggestive of the sporulation of yeasts. On these various and insufficient grounds the independence of the SaccJiaro- mycetes as a group has been questioned, and the suggestion has been made that they are nothing but derivatives of higher groups that have lost the power of repeating the cycle of forms which their ancestors went through. But none of the Saccharomycetes ever show any indications of a higher type of growth ; they produce vegetative cells, cell groups and spores, and never anything else. The announcements that have been made from time to time, that true yeasts have been cultivated from higher FERMENTATION OF SUGARS 131 fungi, have all proved to be erroneous (106). As to the mode of propaga- tion by budding, there is no reason why it should not have appeared independently in several groups, and there are therefore no adequate grounds for refusing the Saccharomycetes the position of a separate self- contained phylum. The Saccharomycetes are mctatrophic and need the same food-stuffs as many bacteria. As a source of nitrogen, peptone is by far the best, and after it asparagine ; but ammonia salts suffice if nothing better is to be had. Carbon is obtained from the fermentable material, sugar, which may be present in quantities up to 35 per cent, (optimum, 2-4 per cent., or 20-25 Pcr cent.). Where the object is not fermentation of the substratum, but culture of the organisms, the sugar may be replaced by other sub- stances, such as glycerine or mannite. In direct contrast to most bacteria, the yeasts are not affected by an acid reaction of the culture medium, but are arrested in their growth if free alkali be present. This peculiarity is of great advantage in brewing and distillery, for by maintaining the acidity of the liquid (wort) in the vats the development of many injurious bacteria is prevented, whilst the yeast cells are unhindered in their fer- mentative power (see p. 120). Of the carbohydrates only the monosaccharides with the formula CcHli;Oc (e.g. glucose, fructose, galactose) are directly fermentable (107). The disaccharides (C12H22OU), cane sugar, maltose and lactose must be in- verted before they can be fermented ; that is to say, they must be changed into monosaccharides (107) *. This is effected, as we have already seen, by the hydrolytic action of enzymes excreted by the yeast cells themselves. By an enzyme called invertase the beer and wine yeasts change cane sugar into the so-called invert sugar, and by another, known as maltose^ they change maltose into glucose. Neither of these yeasts can hydrolyze milk- sugar, and therefore cannot ferment it. This is effected, however, by the Saccharomycetes found in Kephir grains (p. 120) which secrete an enzyme (lactase) that inverts the lactose in milk. Every species of yeast has its own peculiar zymolytic properties. The polysaccharides other than sugars (cellulose, starch, dextrine, gums, &c.) arc not attacked by the yeasts until they have been converted into sugars by enzymes of other origin. In the preparation of beer, for instance, the starch of the barley grains is converted into maltose by an enzyme (diastase) contained in the grains themselves. Every fermentation gives rise, apart from the chief products, to numerous other substances. The following table shows the quantities of these by-products formed in the fermentation of i,coo gr. of grape-sugar. * Probably the various new synthetically-produced sugars will also be nrrangeable in accordance with their structure and ability to be fermented. K 2 132 BACTERIA AND THE CARBON DIOXIDE CYCLE (N. B. — The yeast employed was a wine-yeast, but not a 'pure-culture' according to modern ideas) (108): Grammes. Ethyl Alcohol 506-15 Normal Propyl Alcohol 0-02 I so-butyl Alcohol 0-015 Amyl Alcohol 0-51 Oenanthylic Ether ....... 0-02 Iso-butylene-glycol 1-58 Glycerine . . . . . . . . 21-2 Acetic Acid 2-05 Succinic Acid 4-52 Aldehyde traces In other words, 506 grm. alcohol and about 30 grm. of by-products, to which is to be added about 450 grm. CO2 (estimated). Some one per cent, of the sugar was used by the yeast as food. It is noticeable that as much as two per cent, glycerine is present. This is of some importance, for it has more influence than might be supposed upon the taste of the wine. The two next most important products are acetic and succinic acids. The example just given is of course only one of many different kinds of fermentation ; it must not be taken as a standard. The by-products particularly are very different both as regards quantity and composition in the different technical fermentations. In distillation higher alcohols (e.g. fusel oil) than ethyl alcohol are found. In wine the compound ethers (esters, compounds of alcohols and organic acids) are of prime impor- tance because, although present in homoeopathic quantities, they are the substances which constitute the ' bouquet.' As soon as all the sugar in a fermenting fluid is used up the process ceases, as it does also in any case when the alcohol has reached 12 or 14 per cent. It is from this cause that many Spanish wines remain sweet, alcohol being added to them in order to increase their permanence (?). Alcoholic fermentation (opt. 25-30° ; min. about o° ; max. about 53°) can progress both aerobically and anaerobically. When air has free access to the fermenting fluid the yeast cells multiply with great rapidity, but the power of fermentation, that is to say> the amount of sugar broken tip by unit weight of yeast in unit time, is very small. Conversely, if oxygen be absent or only very sparingly present, the fermentative power of the organisms is great, but the rate of growth very slow. This is best illustrated by a concrete example. To entirely ferment (i.e. to consume all the sugar in) 300 c.c. ' must,' twenty-three days were necessary. The time was the same whether air was present or absent, but the number of cells in each case was very different. Where complete aeration was carried out the fluid contained at the end of fermentation 4,454,800 cells per cubic centimetre, whilst if air was excluded only 50,160 per c.c. were found (109). It is evident that the THEORIES OF FERMENTATION cells in the last case must have been proportionately more powerful in effecting fermentation. All the technical fermentations are, as a matter of fact, anaerobic pro- cesses, for although at first air is contained in the liquids in the vats, and has unhindered access to them, the commencement of the fermentative process alters conditions immediately. The oxygen in the liquid is used up at once by the organisms, and the C(X they evolve lies in a heavy stratum over the contents of the vats, completely shutting off the access of atmospheric oxygen. Under these circumstances the cells arc able to develop their full fermentative power and produce most alcohol. If the object to be attained is not the fermentation of sugar but the growth of the yeast plant, as in establishments for the preparation of ' pressed ' yeast, plentiful aeration must be provided for. TJicory of Fermentation and Putrefaction (HO). The fact that many fermentative processes take place in the absence of oxygen would seem at first sight to give us some clue to the nature of the phenomena. Comparisons have been drawn between fermentation and the so-called ' intramolecular respiration ' of higher organisms. Intramolecular respiration is a term applied by physiologists to the faculty possessed by plants and animals of existing for a short time in an atmosphere devoid of oxygen (pure hydrogen, for example), at the same time giving off CO2 and forming in their tissues a minute quantity of alcohol. It seemed as though all living substance were able to exist for a short time without oxygen, splitting up respirable material (carbohydrates and perhaps albu- mens) just as the Saccharomycetes do. The power of fermenting sugar would indeed be only a special case of intramolecular respiration. We must not however forget that, even apart from purely oxidatory changes such as the acetic fermentation, by no means all fermentative processes are anaerobic. The work of the methane bacteria and of most butyric organisms is indeed strictly anaerobic, but alcoholic fermentation, although most vigorous in the absence of oxygen, is not arrested by the presence of this gas, and most other fermentative processes are similar in nature, i. e. they are facultative anaerobic. The term ' intramolecular respiration ' is intended to indicate a process in which, although C(X is excreted, the oxygen is taken not from the atmosphere, but from the complex molecules of the respirable substance (e. g. sugar), which is broken up into the various less complex bodies we call ' products of fermentation.' That such reductions do in fact take place in anaerobic fermentation (e. g. butyric) may be proved by adding to the fermenting fluid organic colouring-matters such as indigo or methylene blue which become decolourized, that is to say they are converted into the ' leuco ' compound with two additional atoms of hydrogen in the molecule. BACTERIA AND THE CARBON DIOXIDE CYCLE The bleached solutions become blue again if shaken up with air, showing that there has been no far-reaching decomposition of the molecule. All that has happened is that nascent hydrogen has been taken up from the molecules of the fermenting substance. Whether the hydrogen is set free in consequence of the organism seizing oxygen from those molecules, or whether part of it is due to some other disintegration going on in the living protoplasm, is unknown to us. In any case, it is plain that Pasteur's dictum, 'Fermentation is life without oxygen,' does not quite correspond with facts. And the circumstance that fermentation (even alcoholic) can go on in the presence of oxygen is a further argument against such a view, and against the theory that fermentation is a form of intramolecular respiration. Another explanation, older even than Pasteur's, was that proposed by Traube in 1858, the 'enzyme' theory. Traubc imagined that ferment organisms accomplished all their work by means of enzymes excreted by their protoplasm. Such a view could of course be held only as long as it was thought that fermentation was a simple splitting-up of complex molecules into smaller ones : for instance, that alcoholic fermentation took place according to the formula and that no by-products arose. Since we now know that a large number of by-products are formed, and since it has not been possible to isolate the supposed enzymes, Traube's theory had to be abandoned (HI). In one case only, that of the ammoniacal fermentation of urea, did it seem likely that the change was effected by an enzyme, for the process is one of simple hydrolysis (NH2)2CO + 2 H2O = (NH4)2CO3. The enzyme in question, ' urase ' (112), has now been isolated. It is a body of great instability. The difference between the work of enzymes and organized ferments must be here again referred to. Enzymes are doubtless active in all fermentative processes, but the changes they bring about are only prepara- tory ; for example, the inversion of sugars by yeasts, and the peptonizing of albumens by putrefactive bacteria. The actual fermentative processes with their numerous by-products are not to be explained by enzymic action, they are effected by the living protoplasm of the cells *. Another explanation of the phenomena of fermentation, an explanation of a totally different character, was proposed by Nageli in 1879. According to this, the process is entirely extra-cellular, being brought about by vibrations set up by the molecules of the protoplasm and transmitted to the molecules of the substances outside the cell, which are thereby split up into the various bodies we call products of fermentation. This at first sight very plausible hypothesis is rendered extremely improbable by the fact that the cell is * See note No. 111. FERMENTATION AND ANAEROBIOSIS 135 surrounded by a rigid membrane which would at least very much weaken the vibrations if it did not quench them. It is true that the explanation is difficult to disprove, but that is always the case with views based on purely hypothetical molecular movements. The view which perhaps more than any other agrees with the physio- logical or ( biochemical ' nature of the phenomena is that which looks upon them as being the result of chemical processes going on in the living sub- stance of the cells. The fermentative and putrefactive organisms would seem to have properties not possessed by other organisms, properties which enable them to live in places and under circumstances where a complete combustion of the food-stuffs to CO2 and H., O is not possible *. The energy that all other plants and animals gain by respiration is obtained by these organisms from a less complete disintegration of the molecules of the respirable substance, the relative smalhiess of the amount of energy set free being compensated by the large quantity of substance decomposed. In other words, the ordinary respiration of plants and animals is a complete combustion of a few molecules, that of the ferment organisms an incom- plete combustion of many molecules. The products of fermentation there- fore are, unlike the products of respiration, bodies whose heat of combustion is still high ; alcohol, for instance, with 3,246 calories, butyric acid with 3,679 calories. The degree of adaptation (if the expression be allowed) to anaerobic habitats is very different in different organisms. The butyric ferments and the methane bacteria, for instance, are absolutely (obligatory) anaerobic, and have lost the power of ordinary respiration. The alcohol yeasts, lactic bacteria, and most other ferment organisms are only facultatively anaerobic. They are not yet fully weaned from oxygen, which, while not indispensable, is far from being a poison to them. They can, even in the presence of oxygen, exercise their remarkable faculty of partly disintegrating the com- plex molecules of organic compounds, and gaining a small amount of energy thereby. At the same time these facultative anaerobes can actually respire, that is to say, can burn up completely some of the food-stuffs given them. This is proved by the fact that when alcoholic fermentation goes on in the presence of air a larger amount of CO2 is evolved than corresponds to the alcohol formed. Perhaps it is for this reason that the yeasts grow so quickly when air is available ; the extra energy derived from the combustion of the sugar to carbon dioxide and water being used to build up new cell-sub- stance. If oxygen is absent, respiration is not possible ; the other source of energy alone is available, and growth and multiplication arc necessarily slower. * E. g. the mud of ponds and ditches, the interior of decomposing carcases, the contents of the intestine, and below the surface of dung-hills — all places in short where fermentation and putrefaction arc anaerobic. 136 BACTERIA AND THE CARBON DIOXIDE CYCLE This brief description of the phenomena of fermentation will serve to indicate the scope and nature of the problems involved. There remain to be said a few words regarding the by-products of fermentation, carbon dioxide, free hydrogen, and (in putrefactive processes) ammonia and free nitrogen. Careful investigation shows that there is a whole scale of products ranging from compounds whose heat of combus- tion is high, such as alcohol, down to the gases just mentioned, in which it is very low or nil. There seems every reason to believe that the combus- tion of both the chief and the by-products is gradual, by stages. In this way there must be going on a number of different processes simultaneously in the cell, the various compounds being decomposed, and their energy tapped step by step, a process which must necessarily result in the formation of a large number of bodies of different composition — ' by-products.' The description given in the last five chapters of the circulation of the elements carbon and nitrogen, and of the role played therein by bacteria, would be imperfect and inaccurate if no mention were made of the part taken in the process by other micro-organisms. The great importance of bacteria in medicine and in many technical operations has caused them to receive more attention than other groups, and as a result we too often regard them as being the only agents in the disintegration of dead organic matter. This idea is incorrect, for although precise investigations are wanting there can be no doubt that many other protozoa found in abun- dance where putrefaction is going on, ciliate and flagellate infusoria and amoebae, also take part in the work of destruction. They are not always merely saprophile ; they are saprogenic, and assist in the breaking-up of the molecules of organic substance. Nor should we forget that the mould-fungi and all other plants devoid of chlorophyll are entirely dependent for food on decaying organic matter, the carbon of which they help to dissipate by their respiratory processes. They furthermore convert stable and resistant bodies (such as wood) into the easily decayable substance of their own cells. Not a dead twig or trunk in the forest that does not bear minute Pyrenomycetes silently and imperceptibly eating up the wood, to be in their turn disintegrated and dissipated by the putrefactive bacteria to which sooner or later all fungi fall a prey. Thus the final destiny of all living substance is sooner or later, directly or indirectly, to become food for bacteria, and it would seem as though the earth must at last be suffocated by their numbers. But, apart from the fact that large quantities of bacteria are consumed by other protozoa (infusoria and amoebae are often crammed full of them), it must be remembered that each bacterial cell lives only for a short time and leaves but a limited if large number of descendants, which in their turn die off and are consumed by their kindred, only to enter again into new cycles of chemical change. CHAPTER XV BACTERIA IN RELATION TO DISEASE < 1, Diseases of Plants ; Harmless * Messmates ' in the Human Body ; Pathogenic Bacteria ; Points of Attack and Sources of Infection. WITH the exception of the root-nodules of Leguminosae, we know of no single instance where bacteria invade the closed, living cells of plants. The sole and only channels of communication between the interior of the plant and the outside world are the stomata, and these open into a closed system of air-filled intercellular spaces which are shut off from the cells themselves. If bacterial spores are blown by the wind, or washed by the rain into the stomata, they can do no damage, for they can get nowhere but into the intercellular spaces where they find nothing but moist air. Without nutriment, bacterial spores cannot germinate, and nutriment is not present in any form. Even if such microbes as can dissolve cellulose (methane bacteria) drift into the air passages of a plant, they find no food, and are therefore unable to injure the cell-walls. The only organisms whose entrance is accompanied by any danger are those whose spores con- tain sufficient reserve nutriment to enable them to germinate in pure water, to grow at first without food, and open the attack on the cell-wall at their own expense. Such are spores of the parasitic fungi. They contain reserve food-stuffs, and protrude a hyphal filament which pierces the epidermis (potato disease), or pushes its way through one of the stomata into the inter- cellular spaces (rusts], and from thence into the cells themselves. The hyphae of the fungus dissolve the cell-membrane and send in haustoria, or proliferate in mycelial masses in the cell-cavity. Bacteria are totally unable to act in this way, and the uninjured plant is impregnable to their attacks. Even the injured plant offers them nourish- ment only in the exposed and opened cells of the wound-surface, and this source is soon shut off by the development of an impenetrable layer of cork below the wound preventing any further exudation of fluids. Thus the 138 BACTERIA IN RELATION TO DISEASE wound docs not remain moist ; the injured cells shrink and dry up, and the entrance of bacteria is then as difficult as in the case of an uninjured plant. Infected wounds are therefore dangers that have no existence for plants, and the spread of a lesion to other parts is not possible. These considerations enable us to prophesy with considerable certainty what will be the result of an injection into vegetable tissues of bacteria, even of a species pathogenic for animals : no development in the intercellular spaces, and but slight temporary growth on moist wounded surfaces, are what we should expect, and what experiments show to actually occur (113). Notwithstanding these well-known facts, there appear time after time descriptions (generally insufficient enough) of new ' bacterial ' diseases in plants. Bacteria, it is true, arc often found in diseased plants in enormous numbers, but they are living metatrophically only, living on tissues that have already been disintegrated and decayed by parasitic fungi. The bacteria assist these subsequently in their work of destruction no doubt, and modify perhaps more or less the character of the disease. But, leaving aside for the present injuries received from frost or insects, the first attack on the plant is always made by fungi, which are also very harmful in causing the spread of accidental wounds. All the cases of so-called ' bacteriosis' in plants, from the ' goniuwsc bacillairc* of the vine down to the ' scliorf of the potato, arc primarily diseases of non-bacterial origin in which the bacteria are present merely as accidental invaders (114). As metatrophic organisms, bacteria occur plentifully upon the leaves of insectivorous plants such as Pinguicula, Droscra, and Nepenthes. The insect- catching organs are necessarily open to the surrounding world, and bacteria are blown on to them by wind, and brought by insects and other animals. The half-digested remnants of captured organisms furnish a supply of rich nutriment to the bacteria, and it would be extraordinary indeed if they did not flourish and multiply in such places. Here again attempts have been made to demonstrate a ' symbiotic ' relationship between the plant and the bacteria, whose peptonizing powers were supposed to be of service in dis- solving the captured insects. Careful investigation has shown these sup- positions to be incorrect. In the case of LatJiraea, whose leaf-chambers contain hairs often thickly covered with bacteria, it is not certain whether these are metatrophic or not (115). Among the lower animals bacterial diseases have been as yet but little investigated, although they are doubtless very common. The silk- worm diseases discovered by Pasteur, and the so-called ' Foul-brood ' of bees, are well-known examples. Recently a Bacterium ranicidum has been described, pathogenic for frogs and fishes (116). The chief interest as regards disease-producing bacteria is, of course, concentrated on those associated with man and the higher mammalia. The symptomatic characters of bacterial diseases being similar in man n.lCTERLl OF THE MOUTH 139 and other mammalia; the following description will deal only with the phenomena of disease in the human subject. Many bacterial diseases arc common to man and the higher animals, and, as far as we know, there is no parasite of man that is without effect for some animal or another. This fact represents an inestimable advantage in the study of disease, since it enables us, by inoculating animals with pure cultures of bacteria, to put pathological science upon an experimental basis. The whole of our knowledge of micro-organisms in relation to disease, including the promising science of sero-therapeutics, is the direct result of experiments on animals. Bacteria in tJie Hitman Body. — Every human being, even the healthiest, carries about with him innumerable harmless messmates and guests in the shape of metatrophic bacteria. All the cavities of the body that are in communication with the exterior contain them. The intestine, the moist surface of the mucous membrane of the mouth, nose, pharynx, and female genitalia, abound with them (117). They do not penetrate the tissues, but live quite harmlessly in the secretions or excretions, the nature and composition of which determine the character of the bacterial ' flora ' in each particular spot. Some species are constant inhabitants, others only occasional visitors, each kind multiplying and thriving where it finds the most suitable food, and serving perhaps as a protection against the invasion of other and perhaps pathogenic species. Even the relatively dry skin of our bodies always holds spores and vegetative cells. Their character depends largely upon the occupation of the individual, and their number, as might be expected, upon the degree of cleanliness observed. The bacterial flora of the mouth is very rich in species (118). About fifty have been described, some as accidental intruders, others as indigenous organisms. Although never absent, their numbers can be reduced very much by keeping the mouth scrupulously clean. As we have already seen (p. i), their chief forms were known to Leuwcnhoek. Formerly all the bacteria met with in the mouth, whether cocci, rods, vibrios or spirilla, were classed together under one name. They were supposed to be different developmental stages of a single species, Lcpto- tJirix bnccalis, which commonly occurs in the form of long unbranched cell-chains that sprout out from fragments of food, and from the contents of the tonsillar follicles. Such a view is of course incorrect, and the name L. bnccalis must be used only as a collective designation for mouth bacteria. Some of these, such as Bacillus maximns bnccalis (Fig. 26) and a coccus form (lodococcus], give the granulose reaction. Others, such as Vibrio bnccalis (Fig. 26, g}, SpirocJiacte dcntinin (f), and LcptotJirix innominatat stain yellow with iodine. Most of the bacteria of the mouth have as yet resisted all attempts 140 BACTERIA IN RELATION TO DISEASE at culture, and therefore the biochemical characters of the different species arc unknown. This much, however, has been discovered, that the various kinds present, feeding upon the food particles remaining between the teeth, produce lactic and other acids, by which the enamel is decalcified in places. A path to the interior tissues of the tooth being now open, the bacteria bore their way, by means of their acid secretions, deep into the dentinal tubules, just as a lichen eats into stones. The organic substance of the tooth also is destroyed and the tooth becomes hollow and rotten. The following figures illustrate the effect upon the dental tissues : Lime. 187-2 cubic millimetres healthy dentine . 187-2 cubic millimetres carious dentine . . Loss Total Weight. . 0-36 gr. o-26gr. = 72 . o-oS gr. 0-02 gr. = 2 Organic Matter. o-i gr.-28% 0-06 gr. = 74% 0-28 gr. 0-24 gr. 0-04 gr. * til • «', < * -i ill Thus the loss of calcium salts by the teeth caused by the acid secre- tion of the bacteria is 92 per cent., that of the organic substance 40 per cent. Dental caries can hardly be looked upon as a ' disease ' ; it is rather the necessary consequence, sooner or later, of the action of micro-organisms that are normal inhabitants of the buccal cavity introduced daily with the food. The destruction of the teeth is not the work of any one species in particular, but due to the activity of several kinds. Whence the bacteria of the mouth come is not at present known. The typical B. bnccalis viaximus is not found outside the body, and attempts at culture have been unsuccessful. The discovery of bacteria, indistinguishable from those of our own mouths, in the hollow teeth of Egyptian mummies shows that these micro-organisms have been our companions from the earliest times. The healthy stomach is, in consequence of the acidity of the gastric juice, not suitable for the development of a local bacterial flora. If however, as a result of illness or disease, the secretion becomes less acid or neutral, FlG. 26. Bacteria of the mouth and teeth, a, mass of mixed forms (from Miller) ; 6, dentinal tubules rilled and widened by bacteria, partly cocci, partly rods (from Miller) ; c, Spirillum spittigenum ; d, Bacillus maximus bnccalis^ shows granulose reaction ; e, cocci ; f, Spirochaete den- Hum \ f, Vibrio bnccalis ; A, rods, probably lactic acid bacteria (B. acidi lacliri). Magn. a about 250, b 400, c-h about 1 200. BACTERIA OF THE INTESTINE 141 the bacteria and spores introduced with the food thrive and multiply abundantly. The Sarcinae which occur in almost every drinking-water are very prone to make their appearance. They include several species, which were formerly all classed together as S. vctttriculi and supposed to be slightly pathogenic. The richest hotbed for bacteria in the animal body is the intestine. Its contents are alkaline and the prevailing temperature is high and constant, so that putrefactive and fermentative bacteria of all kinds, both aerobic and anaerobic, find optimal conditions of existence. Fresh human faeces contain 75 per cent, water and about i per cent, bacteria. These in- clude spores and rods of all kinds, among which may be often recognized the large cells of B. buccalis maxiimis that have been swallowed with food. It is calculated that from twelve to fifteen milliards of bacteria pass out of the body daily in the faeces (119). Many of them are dead, and stain very badly, others are still alive, and in some spores may be seen. As these spores have not been set free from the rods, there is no doubt they are of recent origin and formed in the intestine. The nature of the putrefactive and fermentative changes wrought by the bacteria on the remnants of undigested food is, of course, dependent largely on the composition of the food. With a meat diet, putrefactive changes predominate, and products like tyrosin, leucin, indol, skatol, sulphuretted hydrogen, and ammonia arise (see Ch. XI). With vegetable nourishment rich in carbohydrates, fermentation takes the upper hand, par- ticularly the methane fermentation of cellulose. If the food and habits of the individual be regular, an intestinal bacterial flora of fairly constant composition arises, the leading species being generally the pleotrophic Bacterium coli comnutne (120) which is both zymogenic and saprogenic (Ch. XVI). Other species (e. g. B.putrificus coli} occur, but have not yet been thoroughly investigated. In the interior of the intestine, towards the middle of the mass that fills it, only anaerobic processes can go on, but at the sides, in contact with the mucous membrane, which itself is thickly covered with bacteria, aerobic changes can occur. It is not necessary to discuss the means by which bacteria obtain entrance into the alimentary canal. We swallow myriads every day with our food, and it is only natural that they should multiply in the very favourable environment that the intestine affords. Their occurrence is a necessary consequence of their wide distribution in nature, and must not be taken as indicating a symbiotic connexion between them and their host. It has been imagined that they were perhaps useful auxiliaries in the process of digestion, but there is no foundation for such a supposition. We are fortunately not so miserably dependent upon bacteria as this. This has been experimentally proved by feeding nc\v-born animals upon sterilized food (121). In this way it is possible to exclude bacteria, and 142 BACTERIA IN RELATION TO DISEASE keep the intestine germ-free for a considerable time, and the animals remain notwithstanding in perfect health. This would certainly not be the case if a symbiotic relationship subsisted between the bacteria and the organism. Such a view is contradicted also by the nature of the products arising from bacterial action. They are quite unfitted for resorption and utilization by the animal body. The intestine of the new-born infant is absolutely sterile (122), but bacteria make their appearance in a very few hours after birth, even before any nourishment is taken. No less than seven different species have been isolated from the intestine of children before feeding. The first to appear is the B. coli commune, which takes up its post at once in every mortal on his entrance into the world, and remains his faithful companion until death. Whence this ever-present species comes is not known, but it is in all probability a metatrophic aquatic form. If infants be fed with the bottle the lactic acid bacteria appear at once in the faeces, and with every new kind of food new species of bacteria are added to the flora of the alimentary tract. As long as the mucous membrane is uninjured these bacteria are quite without danger for the body (123). Once let the continuity of the epithelium be destroyed however, even at a single point, and grave pathological changes may be set up, for among the hosts of metatrophic bacteria inhabiting the intestine some (e. g. B. coli] are facultative parasites and originators of disease. It has long been customary to designate as 'infectious diseases' (124) all those in which the entrance of a certain 'something' into the body was a necessary condition for the existence of the complaint, this 'something,' the pathological 'virus,' playing a part in disease similar to that of the ' ferment ' in the fermentation proce.-ses. Before we knew anything of bacteria it was usual to speak of a contagium when the disease could be transferred only by contact with a diseased individual, and of a miasma when the supposed virus could be carried by the air. And just as the discovery of the ferment organisms displaced the old conceptions of a lifeless ferment, so did the discovery, about the middle of the century, of a ' contagium vivum ' take the place of the old ideas respecting the origin of infectious and epidemic disease. We now know that in the majority of infections the virus animatitin is a bacterium. The demonstration of bacteria in the blood or body fluids of a diseased animal is an easy matter where large species are concerned, such as the anthrax parasite. These can be seen even in the fresh blood as pale rods lying about between the blood-corpuscles ; in this way they were discovered in 1850. More minute forms, particularly cocci, which are easily confounded with albuminous granules from the blood, must be rendered visible by staining with aniline or other dyes. Diseased tissues must be fixed, hardened, cut in sections, and stained in order to render the bacteria in them PATHOGENIC BACTERIA '43 distinguishable from other cellular elements (Fig. 27). Most of the various cytological methods used in investigating cell contents are applicable. The principles of bacteria-staining are not different from those involved in the staining of other cells, although in particular cases special technical pro- cesses are necessary to success. For these any of the numerous handbooks on bacteriological technique may be consulted. The isolation of pathogenic bacteria from diseased organs is effected f X \i ,. c % FlG. 27. Stained preparations of bacteria, from Ziegler's Lehrbuch d. allgem. Pathologie, 8th ed., vol. i. a, sputum of a consumptive, spread on a coverglass, and dried and stained with (uclisin and methylene blue; the tubercle bacilli are red, cellular elements (pus cells) blue. 6, Gonococci (Micrococcits gotwrrhoeae) in fresh secn-tion, coverglass preparation : a, mucus with isolated cocci and pairs ; b and c, pus cells with and without cocci, methylene blue and eosin. c, section through an anthrax pustule (stained by Grain's method), methylene blue and vesuvine. Magn. a 400, b 700, £350. by the method of plate culture (p. 58). In some cases, tuberculosis for instance, the centres of disease contain pure cultures of the specific bacteria, and tubes of nutrient agar or gelatine can be inoculated direct. Much more labour is entailed where the tissues contain a mixture of bacteria of different sorts from which the pathogenic species has to be separated and deter- mined. The requirements of pathogenic bacteria in artificial cultures differ 144 BACTERIA IN RELATION TO DISEASE according to the metatrophic or paratrophic habit of the germ. Strictly- parasitic forms, such as the tubercle bacillus, diphtheria bacillus, and gono- coccus, require the most nourishing media (see Chaps. VI and XVI). Long continued growth in artificial cultures weakens almost all pathogenic bac- teria ; decrease of virulence and morphological changes (involution) showing that nutrient media outside the body can never entirely replace the living tissues. The attenuation can be brought about and increased by many means (see p. 28) and, within certain limits, can be graduated with great precision. It is this possibility of varying the virulence of pathogenic bacteria that underlies a good deal of modern sero-therapeutics and pro- tective inoculation. The fact that the spores and dried vegetable cells of pathogenic bac- teria are in many cases possessed of high powers of resistance renders it possible that the dried secretions and excretions of patients may become a source of infection for the healthy. The tubercle bacillus, for instance, is possessed of its full virulence even after lying for two, or even three, months dried up in dust. As far as the strictly parasitic forms are con- cerned the dry resting-stage is the only form in which they are met with outside the animal body. They have never been found growing and multiplying, for disease products are the only vehicle by means of which they are spread abroad and, once outside the body, even in situations that afford abundant food, they invariably succumb to the rapidly-growing . metatrophic species. But of the many bacteria that are capable of causing pathological changes in the living tissues a large number are of metatrophic habit and able to thrive outside the body. Some grow quickly, others slowly, some have very humble requirements as regards food-stuffs, others are more pretentious, so that the ability to live saprophytically is very different in different cases. All of them, however, represent a much greater danger for mankind than do the obligatory parasites. Not only the pathological products, such as sputum or pus, are possible sources of infection, but all substances, such as milk or impure water, that offer a nutrient sub- stratum and permit these metatrophic forms to multiply, become carriers of disease. The detection and isolation of pathogenic germs from among a mixture of species, such as occur in contaminated drinking-water, is often enough a task of the greatest difficulty, and can be carried out successfully only by those who have had long familiarity in the technical details involved. In many cases experiments on animals are necessary to decide whether a given form is pathogenic or not. Infection of the uninjured body by bacteria most frequently takes place in those organs which communicate with the exterior, particularly in those that, like the respiratory and alimentary tracts, are the passages MODES OF BACTERIAL INFECTION 145 or receptacles for substances taken into the body. They are the natural points of invasion and, as might be expected, they arc furnished with means of defence against it. The epithelium of the stomach and intestine, the mucous membrane of the mouth, and the epidermis of the outer surface of our bodies — in short, every intact epithelial coat — seems to be impenetrable to bacteria. Even when highly virulent germs in large quantities are brought into contact with uninjured epithelium, no infection takes place unless a certain unhealthy condition of the tissues, a ' predisposition,' pre- vails, and for the present this is unexplained. But where there is the smallest break in the continuity of the cells, there the bacteria can penetrate into the tissues and do their deadly work. We see this illustrated every day in wounds on the outer surface of the body, and there can be no doubt that it takes place in the same manner on the inner surfaces, the mucous membranes of the bronchi and intestines. And here it is instructive to recall what happens in plants. These close their wounds from below by impenetrable layers of cork and thus cut off from bacteria the fluids by which they are nourished. In animals, on the contrary, the blood and lymph that exude from a wound form a richly nutritive medium for the growth of the bacteria, which can then penetrate into the tissues and set up ' infec- tion,' localized or general as the case may be. In experiments on animals we imitate the natural course of events by injecting the bacteria beneath the skin or into a blood-vessel. Insect stings represent a natural form of this injection and not infrequently inoculate the body with disease germs. Investigations have yet to be made regarding the number of germs necessary to cause infection, particularly in the case of natural infections. In our experiments on animals the smallest doses must contain thousands of bacterial cells, but it is stated that of the anthrax bacilli ten are enough, injected hypodermically, to cause death in guinea-pigs (125). When bacteria have penetrated below the skin there is always the possibility that they may be taken up by the blood-vessels or the lymphatics and conveyed to other parts of the body. If they are, they may either give rise to local lesions elsewhere or else endanger life by setting up general infection. Anthrax bacilli, for instance, may produce merely a local abscess (malignant pustule), or may spread and multiply in the blood- vessels all over the body. Pus cocci may give rise to little pimples or to the severer form of carbuncle, or scattered abroad by the blood may induce fatal pyaemia or septicaemia. Some bacteria develop most rapidly in the blood (anthrax, spirilla of recurrent fever), others principally or entirely in the tissues (tubercle). The fundamental phenomena underlying all infections are the same although the clinical and pathological features of a disease may be very different in different cases. For these aspects of zymotic complaints medical literature must be consulted. FISCHER L 146 BACTERIA IN RELATION TO DISEASE The relations between the cellular elements of the body and the invading bacteria are probably similar in all cases. Some bacteria are intra- cellular, that is to say, they live in the cell-substance, but the majority are intercellular, the destruction of the cells being a secondary process. The bacteria multiply in the secretions and exudations of the diseased tissue, pushing themselves in between the cells and into the spaces caused by the pathological softening and loosening of connective tissue. CHAPTER XVI BACTERIA IN RELATION TO DISEASE (Continued] 2. Description of some Pathogenic Species. THE following descriptions of some of the more prominent pathogenic bacteria will be confined to the general biological characters of the organism, purely medical questions being left to the specialist, who alone is entitled to discuss them (126). We have seen that among the Saccharomycetes there has been exercised by mankind from the earliest epochs of civilization an unconscious process of selection, which has resulted in the production of innumerable ' races ' or sub-species, distinguishable from each other only by minute physiological differences. A similar process has taken place among pathogenic bacteria. Disease and misery have been the lot of man from the dawn of time, and according to his liability to the attacks of certain species of micro-organisms, and the degree to which they have adapted themselves to life in the tis- sues, races and sub-species have arisen. Differing from one another by physiological peculiarities rather than by morphological characters, these varieties can often only be determined by resort to experiments on animals. In the case of some species that are known only from isolated cases of disease it is impossible even with the most subtle methods to attain certain results, and the difficulties are often increased by the existence of laboratory stocks (see Chap. Ill, p. 28) destitute of hereditary characters. i. COCCI OF SUPPURATIVE PROCESSES (Figs. 28, a-c ; 27,^). By sup- puration is understood the exudation from injured tissues of a fluid filled with wandering cells (leucocytes). It has been experimentally shown that sup- puration can be produced without the agency of micro-organisms — -for instance, by the application of nitrate of silver or corrosive sublimate ; but there is no doubt whatever that all natural suppurative processes, from a sloughing wound down to the tiniest pimple on the face, are caused by micro-organisms. All the commoner pyogenic bacteria are cocci, but in special cases suppuration may be due to other forms, the typhoid bacillus, L 2 148 BACTERIA IN RELATION TO DISEASE for instance, the bacterium of glanders, Actinomyces, and Bacillus pyocyaucus, the bacterium of blue or green pus. The most widely distributed and at the same time most harmless form is StapJiylccoccus pyogcnes aureus (Micrococcns pyogenes). It is a chromogenic species that covers agar with an orange- yellow growth, and sometimes imparts a strong yellow tinge to pus (127). The cells are very small, on an average 0-8 IJL in diameter, colourless, and non-motile. They lie singly, or in pairs, or in short chains ; more commonly still in little clusters (Fig. 28, a}. Besides this orange-yellow form, a paler kind (S. pyogenes citreus) is known, and also a white one (S. p. albus}. They are apparently distinct species, and, although having the same proper- ties as S. anreus, are not so common. In nature the germs are found every- where, so that they would seem to be metatrophic. In disease they are most frequent in local suppurative processes, such as acne (inflammation of the sebaceous glands), sycosis (inflammation of the hair follicles), carbuncle, osteomyelitis, periostitis. Garre rubbed a pure culture of .S. aureus into his arm, and created thereby a severe carbuncle in which the bacteria multiplied abundantly. If from such local foci the bacteria spread, metastatic abscesses arise in other organs and in the joints, and the condition known as pyaemia results. Another common pus bacterium is Streptococcus pyogenes (128). Of this species there are apparently several races very difficult of distinction. Both in the diseased tissues, and in bouillon cultures particularly, it forms long unbranchcd chains with cells somewhat larger than those of StapJiylococcus (Fig. 28, b). The chains result from the planes of division always being parallel to one another. It is regularly present in erysipelas, and in many other pyogenic diseases. It is sometimes associated with StapJiylococcus^ which it exceeds in virulence. Like this, too, it gives rise to pyaemia and septicaemia if carried abroad by the blood. It is often present in diphtheria and in phthisis, increasing the severity of the malady, which then assumes the character of a mixed infection. In cultures, Streptococcus dies off much sooner than does StapJiylococcus ', often in a few weeks. This, and the fact that it is less common outside the body, indicate perhaps that it is a strictly parasitic organism. The bacterium of gonorrhoea, the so-called Gonococcus (129), is cer- tainly of exclusively paratrophic habit. Its artificial culture is only possible upon blood-serum, all other media, however nourishing, being use- less. Whence the Micrococcus gonorrhoeas (Fig. 28, c] originally comes is not known, for it has never been found in nature, and perishes in a few hours when dried up. But it is a constant associate of the human race, and is transmissible only by contact. Since the temperature minimum is about 25° C., and as the cells die within five hours in water, it is evident that a multiplication of the organism in swimming-baths is not possible. There is no danger of infection from this source. The gonococcus occurs in the inflam- ANTHRAX 149 matory secretion of the urethra, both in the cells (Fig. 27, /;) and in the fluid. Thence it spreads to the epithelium and mucous glands, and finally to the other parts of the genital system. It may even extend all over the body (gonorrhocal rheumatism). The cocci lie usually in pairs, the stained cells being separated by a clear unstained line where the mutually flattened sides are in contact. They are non-motile, and about the same size as staphylo- cocci, from which, however, they are readily distinguishable by the paired grouping of the cells. None of the above-mentioned cocci are known to form spores. A not infrequent cause of inflammation and suppuration in human beings is the ' Pneumococcus ' (Diplococcns pnenuwniae} of Frankel, the usual cause of pneumonia (130). 2. ANTHRAX (131) (Figs. 28, d; 27, c\ 5,^,7; u, <*•£•; 29)- The anthrax bacillus (B. antJiracis] was discovered about the middle of the pre- sent century (in the early fifties) in the blood of cattle suffering from splenic fever. It was detected in the form of colourless, motionless rods lying about between the blood-corpuscles. Although from the first suspected to be the morbid agent, proof was not available until many years afterwards (1863). Through Koch's investigations into the life-history of the organism, anthrax or splenic fever has found its way into all the text-books as a clas- sical example of a bacterial infection. Koch followed the process of spore formation, and obtained the first pure cultures of the bacillus, commencing with this work his brilliant career as the founder of modern bacteriological technique. The anthrax parasite is a comparatively large bacillus, cylindrical in shape, 3-6 \t. long by 1-5 \j, thick, varying within certain limits, as all bac- teria do. Both in the blood and in the tissues single cells and cell-chains occur (Fig. 27, c\ In cultures the growth is chiefly filamentous, the plate colonies being as a result fringed with shaggy edges, loops, and curls of filaments stretching out into the gelatine. For the same reason stab cul- tures have a bristly appearance. The cells are non-motile, but form spores readily *. The anthrax bacillus grows well in artificial cultures, but needs good sources of nitrogen and carbon ; it is a peptone bacterium. There is, however, no doubt that it is not an obligatory parasite, but metatrophic, for it has been observed to grow well and form spores in cow-dung and earth. This indicates how the disease may arise in cattle, which are much more subject to infection than human beings. In man, infection generally takes place from skin wounds, and the disease remains localized, rarely spreading to other parts. In cattle, on the other hand, it is with food that the * \\ith regard to spore formation, see p. 20; germination, p. 22. ; resistance to beat, p. f dryncss, p. 77 ; poisons, p. 82 ; degeneration and attenuation, p. 28. 150 BACTERIA IN RELATION TO DISEASE organism (probably spores, for the most part) is taken up. The spores pass uninjured through the stomach, and germinate in the intestine, setting up intestinal anthrax, which mostly leads to general infection and death. It is not known whether the bacteria have themselves the power of boring through the uninjured mucous membrane of the gut, or whether a previous laceration is necessary, such as might be caused by jagged splinters of food. In small animals, such as mice, which succumb very quickly (1-3 days), the disease causes no conspicuous change in the organs, but in sheep and cattle its effects are more conspicuous. Spores, being formed only where there is free access of oxygen, never occur in the diseased tissues or the dead body. They arise under suitable conditions of temperature (i8°-3O° C.) in the bloody dejecta of sick animals, and also in carcases that arc not buried deeply underground. 3. TETANUS (132) (Fig. 28, e). Lock-jaw or tetanus is caused by a metatrophic bacterium very widely distributed in the soil, where it probably gives rise to various putrefactive and fermentative processes. The nature of these processes is in all probability dependent upon the food that happens to be available, for the tetanus organism is able both to split up sugar, and in albuminous substances free from sugar to decompose protcids into CO2, SH2, CH4, mercaptan, and free hydrogen. The pathogenic properties of the organism are due, not to any of these substances, but to a violent poison which has not yet been isolated in a perfectly pure form. Tetanus is a typical traumatic infection that arises only by the contamination of wounds by matter containing the bacillus — earth or the dust of hay or straw, for example. The bacilli grow only in the wound itself, and even here but sparingly. They never spread to other parts of the body. Bacillus (Plectridium) tetani is a slender motile rod 2.4^1 long by 0-3 — 0-5 M thick. It is inclined to filamentous growth, particularly in anaerobic cultures. In aerobic cultures the moss-like colonies grow only in the deeper layers of the gelatine. Before sporulation, which is very constant, the rods swell up at one end, and in this dilated part the spore appears. The drum- stick shape, together with the peritrichous type of ciliation, places the organism in the genus Plectridium. Two other bacteria of the soil with saprogenic and zymogenic proper- ties (B. Chauvei and B. oedematis maligni) give rise to quarter-evil and malignant oedema respectively. 4. DIPHTHERIA (Bacillus diphtheriae, Loeffler; Corynebacterium diphtJieriac, Lehmann and Neumann [133]; Figs. 28, f and 14, /i). This bacterium is found almost without exception in the outer layers of the diph- theritic false membrane. In consequence of this tendency to superficial growth, it seldom spreads to other parts of the body, but remains confined to those cavities which are the usual seat of the disease. Exceptions to this do, however, sometimes occur. It is often found in the throat in association DIPHTHERIA 151 with Streptococci, and in some few cases of apparently indisputable diph- theria it has seemingly been wanting. It is a true parasite, and in cultures requires the very best nutriment. Growing best upon blood-scrum, to which has been added bouillon and sugar, it shows a tendency even on this medium to produce involution forms, although it multiplies rapidly. Irregularly swollen rods arise, and even cells with short branchings (Fig. 27). As was already mentioned, these have been without sufficient reason looked upon as representing a higher morphological stage than the normal rods. In the diphtheritic membrane, and in fresh cultures, the bacillus appears as a minute, somewhat clubbed or oval, rod-like cell about 1-5-2 p. long by 0-5 fj. thick. It is non-motile, and spores are unknown. In young cultures the cell-contents stain apparently uniformly. Not infrequently strongly-tinged granules can be seen, and these when they are large and lie at the ends of the cell give the impression of spores. They are, however, nothing but ' chromatin ' granules, such as have been already described. The apparently uniformly-stained cell-contents have the same structure FIG. 28. Pathogenic bacteria, a, Stapltylococctis pyogenes atireiis (Micrococcus pyogenes) ; f>, Streptococcus pyngenes', c, Micrococcus gonorrhoeas; . (p. 161). METSCHNIKOFF, Ueber die Bczichungcn der Phagocylen zu Milzbrand- bazillen, Virchow's Archiv, xcvii. 1884, Thcoric des Phagocytes, Annales Pasteur, i. 1887, and numberless other papers by METSCHNIKOFF that are largely devoted to the polemical war that arose over his doctrines, such as : FLIJGGE, Studien iibcr die Abschwachung virtilentcr Bakterien und die erwor- benc Immunitiit ; BITTER, Kritische Bcmcrkungcn ?.u Mctschnikoffs Phago- cytenlehre ; NUTTALL, Experimente iiber den baktericnfeindlichen Kinfluss des ticrischcn Korpcrs, Zeitschrift f. Hygiene, iv. 1888; BAUMGARTEN, Ueber das 1 Experimentum crucis' dcr Phagocytenlehre, Zicgler's Beitrage zur patliol. Anat., vii. 1889 —METSCHNIKOFF, Immunitiit, in Weyl's Handb. d. Hygiene, vol. ix., i. Lief. 1897. 14'.i. (p. 162). BUCHNER, Ueber die bakterientotende Wirkung des zdlenfreien Blut- serums, Centralbl. f. Bakt. v. and vi. 1889 ; Ueber die nahere Natur dcr bakterientotenden Substanz im Blutserum, ibid. vi. ; Untersuchungen iiber die bakterienfeindlichen Wirkungen des Blutes und Blutserums, Archiv f. Hygiene, x. pp. 84-173, 1890. FODOR, Neue Untersuchungen iiber die bakterientotende Wirkungdes Blutes und iiber Immunisation, Centralbl. f. Bakt. vii. 1890. See also notes 154 to 161 on the specific serum reactions. 147. (p. 163). BORDET, Sur le mode d'action des scrums prcVentifs, Annales Pasteur, 1896, x. ; Les leucocytes et les propriete's actives du serum chez les vaccine's, ibid. ix. 1895. Roux, Sur les serums antitoxiques, ibid. 1894, viii. HAHN, Ueber die Beziehungen der Leukocyten zur baktericiden Wirkung des Blutes, Archiv f. Hygiene, xxv. 1895. 148. (p. 163). KOBERT, Lehrbuch der Intoxikationen, 1893, pp. 261, 554 ; EHRLICH, P., Experimentelle Untersuchungen iiber Immunitat, Deutsche mediz. Wochenschr. 1891, Nos. 32 and 44 (experiments with ricin and with abrin, the poisonous principle in the seeds of Abnes precatorius}. 149. (p. 163). BEHRING, Infektion und Desinfektion, 1894, p. 172, &c., and many other papers ; see also the subsequent notes. EHRLICH, KOSSEL, and WASSERMANN, Ueber Gesvinnung und Verwendung des Diphtherieheilserums, Deutsche mediz. \Vochenschr. 1894, No. 16; EHRLICH and WASSERMANN, Zeitschr. f. Hygiene, xviii. 1894. 150. (p. 164). BEHRING, Die Blutscrumtherapie. i. Die praktischen Zicle und die Immunisierungsrnethoden zum Zweck der Gewinnung von Heilserum ; ii. Das Tetanusheilscrum und seine Anwendung auf tetanuskranke Menschen. Leipzig, 1892. 151. (p. 164). Roux and MARTIN, Contribution a 1'e'tude de la diphtheric, Annales Pasteur, viii. 1894. 152. (p. 164). In BEHRING, Bekampfung der Infektionskrankheiten, Infektion und Des- infektion, 1894, p. 1 88, and in many other passages, the specific antitoxic effect of the serum is emphasized. It has no anti-bacterial (bactericidal) action. 153. (pp. 161, 165). EHRLICH and WASSERMANN, Ueber die Gewinnung der Diphtherie- antitoxine aus Blutserum und Milch immunisierter Tiere, Zeitschr. f. Hygiene, xviii. 1894. 154. (p. 165). EHRLICH and HUBENER, Ueber die Vererbung der Immunitat bei Tetanus, Zeitschr. fur Hygiene, xviii. 1894. VAILLARD, Sur rht'reditc de 1'immunite acquise, Annales Pasteur, x. 1895. 184 NOTES 155. (p. 165). BEHRING, Infektion und Desinfektion, p. 160, and Deutsche mediz. Wochenschr. 1893, No. 48. WLADIMIROFF, Ueber die antitoxinerzeugende und immunisierende Wirkung des Tetanusgiftes, Zeitschr. f. Hygiene, xv. 1893. 155. (p. 165). If this assumption were correct it would explain the well-known fact that only the earliest stages of diphtheria can be successfully treated by the serum method. It would account too for Koch's observation (note 143) that the new tuberculin T. R. must be used within one or two weeks of inoculation if it is to have a really curative effect. In all these cases the toxines introduced artificially may be imagined to bring about toleration before the body is inundated with poison from the bacterial centres. 157. (p. 166). Roux, Sur les serums antitoxiques (Ann. Pasteur 1894), thinks it probable that the antitoxines act upon the tissue cells and make them insensitive to the toxines. BEHRING (Infektion und Desinfektion) is inclined to think the toxines are destroyed by the antitoxines. 158. (p. 166). BEHRING in the works cited ; also in Die Geschichte der Diphtheric, Leipzig 1893, and Gesammelte Abhandlungen zur atiologischen Therapie, Leipzig 1893. EHRLICH, Die staatliche Kontrolle des Diphtherieheilserums, Berl. klin. Wochenschr. 1896. 159. (p. 166). Of the innumerable works on the value of serum therapeutics that have already appeared may be mentioned : BEHRING, Die Statistik der Heilserum- frage, Marburg 1895 ; HEUBNER, Klinische Studien iiber die Behandlung der Diphtherie mit dem Behring'schen Heilserum, Leipzig 1895 ; ESCHERICH, Diphtheric, Croup und Serumtherapie, 1895 ; GOTTSTEIN and SCHLEICH, Immunitat, Infektionstheorie und Diphtherieserum, Berlin 1894; GANGHOFNER, Die Serumbehandlung der Diphtherie, Jena 1897. 160. (p. 166). PFEIFFER, Die Differentialdiagnose der Vibrionen der Cholera asiatica mit Hilfe der Immunisierung, Zeitschr. f. Hygiene, xix. 1895; PFEIFFER, Centralbl. f. Bakt., xix. 1896, pp. 191, 385, 593; ibid. xx. 1896, p. 129. BORDET, Sur le mode d'action des serums pre'ventifs, Annales Pasteur, 1896. DUNBAR, Zur Differentialdiagnose der Choleravibrionen, Zeitschr. f. Hygiene, vol. xxi. 161. (p. 167). PFEIFFER and PROSKAUER, Beitragezur Kenntnis derspezifisch wirksamen Korper im Blutserum von choleraimmunen Tieren, Centralbl. f. Bakt., xix. 1896, p. 197. 162. (p. 168). PASTEUR, Comptes rendus, Oct. 26, 1885, and in many other papers on this very remarkable inoculation, which is indeed a kind of serum treatment. For the organs employed (cord and brain) contain not only the postulated antitoxin, but also the poison which is thus introduced into the bitten animals, only by a different vehicle. Pasteur's genius did not need pure cultures of the unknown hydrophobia germ, and this gives us a right to hope that we may succeed with his method in the case of other diseases. 163. (p. 168). CHAMBERLAND, Resultats pratiques des vaccinations contre le charbon et le rouget en France, Annales Pasteur, viii. 1894; see also note 17. 164. (p. 168). GRUBER, Miinchener mediz. Wochenschr. 1896, applies the term ' glabri- ficines' to the substances in the blood of immunized animals, which cause the membranes of the bacteria to swell up, and the rods themselves to agglutinate. 4 Lysines ' and 'antilysines' were introduced by KRUSSE (FLUGGE, Mikroorg., 3rd ed., vol. i. pp. 409, 414). Of all these substances we know only the names. INDEX Abeles, criticism of Huchncr, p. 179. Acetic acid, 100; production of by anthrax bacilli in milk, 118, by bacteria, 113, 114; by butyric — ,121 ; in fermentation of bread, 125; by kephir, 120; by yeast, 132. Acetic bacteria, in, Fig. 24; literature of, 176; cessation of growth of in partial vacuum, 61. Acetic fermentation, 112; activity of, 113; equation for, 112. Achorion Schoenbeinii, occurrence of in Favus, 42. Acne, cause of, 148. Actinomyces, suppuration induced by, 148; A. bovts, culture and characters of, 41 ; supposed sporangia of, 42. Aerobes, definition of, 60. Aetherial oils as disinfectants, 86. Agar media, 57 ; non-liquefaction of by bacteria, 59. A}r, presence of bacteria in, 44. Albumoses, properties of, 99. Alcohol, heat of combustion of, 135 ; in- hibitory percentage of, 83 ; - for acetic bacteria, 112; lethal percentage of, 83 ; resistance of spores to, 84 ; negative chemotaxis of, 79. Alcoholic fermentation, 126 seq., 178, 131 ; by-products of, 132; influence of oxygen and temperature on, 132. Alcohols, nutritive value of, 57 ; fermenta- tion of, 114. Aldehydes, production of by yeast, 132. Alder, root-tubercles of, 89. Alexines, occurrence and importance of, 162. Alimentary canal, bacteria of, 180. Alkalies, influence of on toxines, 160; negative chemotaxis of, 79. Allococcaceae, characters of, 32, 33. Alvarez, on indigo fermentation, 178. Amido-bacteria, 55 ; -- compounds, 99. Amines, nutritive value of, 57. Ammonia, occurrence of in cheese, 119; production of by putrefaction and fer- mentation, 103. Ammonia-bacteria, 55. Ammonium chloride, as source of nitrogen, 55 ; — tartrate as source of N, 55. 'Amoeba, importance of, 39; rapidity of fission in, 18; A. colt, connexion of with dysentery, 40. Amygdalic acid, bacterial decomposition of into + and - optical components, 115. Amyl alcohol, production of by yeast, 132. Amylobacter butyricus of van Tieghem, 121. Anaerobes, definition of, 60. Anaerobic bacteria, distribution and import- ance of, 61 ; — fermentation, reduction of indigo during, 133. Anaerobiosis, literature of, 172, 176 ; rela- tion of to fermentation, 133, 135. Anilin dyes, as disinfectants, 86. Animalcula, definition of, I. Anthrax bacilli, abscesses due to, 145 ; aspo- rogenous varieties of, 170; facultative parasitism of, 49 ; formation of chains by> 3! germs of, 149; growth of in fluid media, 58; in milk, 117, 118; history of, 149; infection by, 145; inoculation for, 168; literature of, 181; transference of, 150; — , inhibitory per cent, of poisons for, 82; --lethal per cent, for spores of, 83 ; permanence of, 30 ; plate and stab-cultures of, 59 ; resistance of to low temperatures, 75 ; size of, 4 ; — spores, vitality of, 77. Antilysines, 168, 184. Antiseptics, 173; — treatment of wounds, .87-. Antitoxins, hypothetical character of, 166; literature of, 183, 184; occurrence and properties of, 162, 163 ; theory of, 165. Aphanothece, shape of, 37. Arsenious acid, influence of on ferments, 109. i86 Arthrosporcs, 22, 25. Aseptic treatment of wounds, 87. Ash, percentage of in bacteria, 52 ; essential amounts of, 53, 54. Asparagin, as source of nitrogen, 55 ; chemotactic attraction of, 79. Aspartic acid, 99. Asp&rgillus, parasitism of, 42; patho- genicity of species of, 42 ; -- yeast, 1 10, 176. Asporogenous condition, induction of, 28. Attenuation, 27 ; influence of on spore formation and virulence, 28. Australia, nitrite bacteria of, 105. Autoclave, use of, 76. Bacilleae, 33. Bacillus, I ; definition of, 2, 32 ; characters of genus, 33; B. a'cti, in, Fig. 24, action of I on, 113; B. (nidi I act id, in, Fig. 24, shape and properties of, 117; B. acidificans longissimus, shape of, 117; B. aerogencs, shape of, 117 B. ant /tract's, capsules of, 10, Fig. 7 characters and life-history of, 149 formation of spores of, 20, Fig. 1 1 conditions for--, 23, 150; germina- tion of, 20, Fig. II, 22; growth of in culture media, 55 ; plugging of capillaries by, 159; production of caprionic acid in milk by, 1 18 ; tempera- ture optimum for, 28, minimum and maximum for, 74 ; B. brunneus, colour of, 12 ; B, bttccalis maximits, p. I, Fig. i; granulose reaction of, 139; shape of, 140, Fig. 26; occurrence of in faeces, 141 ; B. Chauveii, 150, pathogenic character of, 112; />'. coli, 101, 151, Fig. 28; growth of on different culture media, 55 ; influence of acidity of on, 56 ; non-liquefaction of gelatine by, 58 ; production of indol by, 102; B. coli connnunis, 15; fer- mentative properties of, 112; fermenta- tion of carbohydrates by, 102 ; in- fluence of peptone and ammonia on fermentative products of, 115; B. cyaneo-fitscits, pigment of, 13 ; B.cyano- genus, colour of, 12 ; discolouration of milk by, 118; B. diphtherias, 151, Fig. 28; characters of, 150, 151, Fig. 28 ; B. ethacetictis, fermentative activity of, 114; B. fluorescens lique- faciens, chemotactic attraction of, 79, Fig. 18; putrefactive properties of, 1 02 ; B. indigogemis, fermentation of indican by, 124 ; B. Kiitzingianus, in, Fig. 24 ; "action of I on, 112; B. lepto- sportis, germination of spore of, 20, Fig. n, 22; B. maligni oedematis, 150; B. orthobutylicus, fermentative activity of, 114; properties of, 121; B. Pasteurianus, ni, Fig. 24; action of I on, 112; fermentative activity of, 113; B. phosphorescens, minimum, maximum, and optimum temperatures for, 74 ; B. prodigiosus, colour of, 12 ; coloration of milk by, 118; percentage composition of, 52 ; production of lactic acid by, 116; B. protcus, 15, 102, Fig. 22 ; B. pittrificus coli, occurrence of in intestine, 141 ; putre- factive products of, 102 ; B.pyocyaneus, decomposition of fat by, 108 ; growth of on culture media, 55 ; in sugar solution, 56 ; suppuration due to, 148; B, radicicola of Beyerinck, 92 ; B. snbtilis, 15, Fig. 8 ; cessation of growth of in partial vacuum, 61 ; ciliation of, 15; permanence of — , 31 ; supposed conversion of anthrax bacilli into, 30; germination of spore of, 20, Fig. 11, 22 ; growth of on culture media, 55 ; influence of acidity of on, 56 ; in- volution forms of, 26, 27, Fig. 14; life cycle of, 25, 26, Fig. 13; minimum, optimum, and maximum temperatures for, 74; rapiJity of fission in, 17; resistance of spores of to boiling, 76 ; B.tetani, 151, Fig. 28; characters of, 150; obligate anaerobiosis of, 61 ; B. thermophilus, optimum, minimum, and maximum temperatures for, 74 ; />'. tuberculosis, 151, Fig. 28; absence of spores in, 153; characters of, 153; culture of, 152; cellulose in cell-wall of, 9; growth of in fluid media, 58; minimum, maximum, and optimum temperatures for, 74 ; tingibility of, 181 ; B. typhi, 151, Fig. 28 ; characters of, 154; ciliation of, 15; growth of in culture media, 55 ; non-liquefaction of gelatine by, 58 ; polar granules of, 9; B. typhi murium, use of, 154; B. ureae, 102, Fig. 22 ; B. violncctts, arthrospores of, 23 ; colour of, 12; distribution of pigment in, 13 ; in- fluence of oxygen on pigment formation of, 61 ; B, wrens, distribution of pig- ment in, 12; B. I'iscosus sacchari, nutrition of, 123 ; B, vulgaris, nutritive requirements of, 29 ; putrefactive powers of, 102 ; zoogloea of, 31. Bacteria, of mouth and teeth, 140, Fig. 26; diseases of plants induced by, 138 ; non-penetration of plants by, 138. Bactericidal substances, 83, 166. Bacteriological analysis, methods and technics of, 171 ; of soils, 47. Bacterio-purpurin, functions and spectrum of, 68, 69. Bacteriosis, 138. Bacterium, definition of, 32 ; dimensions of, 4 : B. accticum, 1 10 ; />. acidi INDEX 187 1,-iLlici, definition of, 116; />'. bu'yri- itiiH, 1 10 ; />'. <•('//' Ciwiinun ; occurrence and properties of. 141, 142; B. Pttsfeurianum, involution forms of, 27, Fig. 14; B+pkotometricwri) photo- p'.iilism of, 72 ; B, ranicidum, patho- genic character of, 38; B. tcrmo, definition of, lot ; B. Zopfii, 102, Fig. 22. Bacteroids, as involution forms, 27 ; shape of, 90, Fig. 19. Bactridium^ characters of genus, 33 ; B. coli, 151, Fig. 28; I>. cnli commune, 154; B. proteus, 1 02, Fig. 22 ; colony of, 4; H. typhi, 151, Fig. 28. Bactrillum, characters of genus, 33 ; B. pseudotermo, 102, Fig. 22. Bactrinium, characters of genus of, 33. Baier, on butyric fermentation, 177. Baktron, 32. Barium, substitution of for calcium, 54. Baumgarten, on phagocytosis, 183 ; on pathological mycology, 1 80. Beer, mucilaginous 'fermentation' of, 123 ; spoiling of, 1 20 ; - - yeast, purification of, 120. Bees, bacterial disease of, 138. Beif^iatoa, 65, Fig. 17, 66 ; characters of, 2, 34; oscillating movements of, 16. Behrens, on tobacco fermentation, 178. Beh ring, on antitoxic serum, 184; on dis- infection, 173, 184; on immunization of horses to tetanus, 164 ; on infection, 100 ; on serum therapeutics, 183. Behring and Ehrlich, on antitoxins, 166- Benzoic acid, nutritive value of, 57. Bernheim and Folger, on involution forms of diphtheria bacilli, 170. Beyerinck, on artificial culture of root- tubercle bacteria, 92 ; on assimilation of nitrogen by Bacillus radicicola, 174 ; on butyric bacteria, 121 ; on butyric fermentation, 177; on glucose, 178; on nutrition of bacteria, 56, 171 ; on pigment bacteria, 169; on phosphor- escent bacteria, 172; on root-tubercle bacteria, 1 74 ; on Spirillum dcsulfuri- ctins, 176. Bienstock, on faecal bacteria, 175. Bile, action of on bacteria, 87. Billroth, on bacteria of wounds, 28 ; on species of bacteria, 170. Biochemistry, Chap. X-XIV. Bitter, on phagocytosis, 183. Blastomycetes, 126. Blood, occurrence of bacteria in, 142; pathogenic bacteria of, 145. Blood-serum, as nutritive medium, 57. Bordet, on preventive serums, 183, 184. Botrytis cinerea, inhibitory action of light on spore formation in, 72 ; rotting of fruits due to, 99. Bouquet of wine, origin of, 129. Branching, of filamentous bacteria, 3. Brancll, on anthrax, 81. Bread, cause of rising of, 125. Brefeld, on division of bacteria, 170; on fungal yeasts, 178; on germination of bacterial spores, 170; influence of lig In on fungi, 172. Bricger, on ptomaines, 100, 175; B., Boer and Colin, on tetanus poisons, 182 ; B. and Friinkel, on bacterial poisons, 182. Bromine water, lethal percentage of, 83, 84. Brown, on fermentative power, 179. Brownian movement, 14. Bruns, on tubercle bacillus, 170. Buchner, on alexines, 162 ; on bacteri- cidal action of blood serum, 183 ; on chemotaxis of leucocytes, 173; influence of light on bacteria, 172; Ed. Buchner on zymase, 179; H. Buchner, on alcoholic fermentation without yeast cells, 179. Buchner, Langard, and Riedlin, on rapidity of multiplication of bacteria, 170. Budding, rapidity of in yeast cells, 126. Bumm, on micro-organisms of gonorrhoea, 181. Burri and Stutzer, on denitrifying bacteria, 175- Biisgen, on culture of CladotJirix dichotoma, 170. Biitschli, on Cyanophyceae and Bacteria, 169. Butter, number of bacteria in, 118; causes of rancidity and of flavour of, 1 18. Butyl-alcohol, production of by bacteria, 114, 122. Butyric acid, 100; heat of combustion of, 135; occurrence of in cheese, 119; production of by bacteria, 114; --in rancid butter, 118. Butyric bacteria, fermentation of lactic acid and of milk by, 122; spoiling of beer by, 120 ; varied nutrition of, 29. Butyric fermentation, 121 ; literature of, 177 ; products of, 122. Cadaverine, 100; inhibitory percentage of, 82. Cadeac and Bournay, on action of diges- tive fluids on bacteria, 174. Caesalpineae, occurrence of root-tubercles in, 91. Caesium, substitution of for potassium, 54. Calcium lactate, fermentation of, 122. Cancer, supposed origin of, 39. Cane sugar, inversion of, no. Capsules, occurrence of, 10. i88 IXDEX Carbohydrateb, bacterial fermentation of, 1 16 seq.j function and occurrence of, 53- Carbolic acid, inhibitory percentage of, 82 ; lethal — of, 83. Carbon, sources of, 55 ; —compounds, nutri- tive importance of, 56, 57 ; - - dioxide, circulation of, 107, chap. XII ; inhibi- tory action of, 86 ; - - monoxide, in- hibitory action of, 86. Carbuncle, causes of, 145 ; production of by pus bacteria, 148. Caries of teeth, causes of, 140. Casein, in cheese, 119 ; in curds, 119. Caseoglutin, occurrence of in cheese, 119. Cattle, anthrax in, 149, 150. Cell-wall of bacteria, attachment of, 8 ; cellulose in, 9 ; chemical composition of, 9 ; permeability of, 8, 9. Cellulose, fermentation of, 122, 177 ; occur- rence of in tubercle bacilli, 153. Certes, influence of high pressures on bacteria, 173. Chamberland, on attenuation, 170; on preventive inoculation, 184. Cheese, bacteria of, in, nS; composition and ripening of, 119; literature of, 177. Chemical composition of bacteria, 52. Chemotaxis, 78 seq., chap. IX ; literature of, 173 ; application of Weber's law to, 80 ; character of movement and in- fluence of concentration, Si ; negative, 79 ; operation of in nature, 80. Chilian saltpetre, origin of, 104. Chlorine, necessity of, 54 ; — water, lethal percentage of, 83, 84. Chloroform, action of on ferments, 109. Cholera, germs of, 155 ; — decomposition of fat by, 108 ; facultative parasitism of, 49 ; growth of in fluid media, 58 ; - in plate cultures, 59 ; — in milk, 1 1 7, 118; literature of, 182; polytrophism of, 29; production of indol by, 101 ; reproductive activity of, 17 ; transfer- ence of, 156; -- serum, 167. Chromatin granules, occurrence of in diphtheria bacilli, 151 ; in Cladothrix, 7, Fig. 5- Chromattum, 66 ; distribution of pigment in, 12; C. Okenii, 65, Fig. 17. Chromic acid, as fixing fluid, 6. Chromogenic bacteria, 12. Chromoparous bacteria, 12, 13. Chromophorous bacteria, 12. Chroococcus, shape of, 37. Cider, influence of bacteria on, 114. Cilia, abscission of, 15 ; anomalies in con- nexion with, 1 8 ; influence of external agencies on, 16 ; constancy and importance of, 31 ; forms of, 15, Fig. 8; influence of oxygen on, 61 ; use and staining of, 14. Ciliation, taxonomic importance of, 15. Citric acid, production of by mould fungi, no. Cladonia furccda, 93, Fig. 20. Cladophord) evolution of oxygen from, 62, Fig. 16; C. fracla, process of cell- division in, 17. Cladothrix, 36 ; arthrospores of, 22 ; charac- ters of, 34 ; occurrence of iron in, 70 ; sap-vacuoles in, 6 ; sliding growth of, 18; sheaths of, 10; C. dicliotoma, 3, Fig. 2 ; chromatin granules of, 7. Fig. 5 ; formation of swarm spores by, 25, Fig. 12 ; pleomorphy of, 24. Classification of bacteria, 32, 33, 34 ; difficulty of, 30; features of, 30, 31. Claudon and Morin, 178. Clostrideae, 33. Clostridiitni, characters of, forms of, 121 ; C. butyricuin, 1 1 1, Fig. 24, 121 ; forma- tion of spores of, 20, Fig. 1 1 ; germina- tion of, 22 ; C. Pasteurianum, charac- ters of, 96 ; fixation of N by, 96. Coal gas, inhibitory action of, 86. Coccaceae, 32. Cocci of suppuration, 147, 148, 149. Cociiditi, 41. Coccobacteria septica, 29. Coccus, definition of, 2. Cohn, 2 ; on B. termo, 101 ; on classification of bacteria, 29, 30, 31, 171 ; on thermo- genic bacteria, 63, 172 ; on putrefactive bacteria, 175 ; on relation of tempera- ture to bacteria, 173 ; on resistance to desiccation, 173 ; on species of bacteria, 170; on spores of, 170. Cohn and Mendelssohn, influence of electric currents on bacteria, 172. Colloid silica media, 58. Colon bacillus, 154. Comma bacillus, 156; shape of, 2. Concentration, influence of on bacteria, 9. Conn, on influence of bacteria on flavour of butter, 177. Contagium animatum, 159; - - vivum, 142. Coppen Jones, on branching of tubercle bacilli, 170. Coprinus, influence of light on, 72. Cornil and Babes, 180. Corrosive sublimate, poisonous action of, 57- Coryne bacterium, 27 ; C. diphthenae, 1 50, 151. Cow-pox, microbe of, 40. Cramer, on composition of bacteria, 171. Crenothrix, characters of, 34 ; sheaths of, 10 ; iron in, 70. Culture, general principles of, 52, chap. VI ; - media, 54, 55 ; -- fluid, 56 ; growth in, 58 ; for phosphorescent bacteria, 63 ; - - solid, use of, 57, 58; plate, stab and streak, 58, 59 ; influence of on per- INDEX 189 centage composition, 52 ; --of acidity and alkalinity of, 55, 56, 131. Cupric sulphate, inefficiency of as disin- fectant, 84 ; lethal percentage of, 83. Curds, composition of, 119; influence of bacteria on, i 19. Cyanin, inhibitory percentage of, 82. Cyanogen, as source of carbon, 57. Cyanophyceae and bacteria, relationship between, 37. Cytoryctcs I'ariolae, association of with cow- pox, 40. Czaplewski, on tubercle bacilli, I Si. Davaine, on anthrax inoculation, 181. Dawson, on efficacy of nitragin, 93. De Bary, on arthrospores, 22, 170; on species in bacteria, i/o ; on systematic position of yeast, 178. Denitrifying bacteria, 176. Desiccation, influence of on maximal temperatures, 76; resistance of bacteria to, 19, 77, 144, 173; of spores to, 19, 44,45, 128. Dextrane, occurrence of in slime bacteria, 53- Diastase, hydrolytic action of, 109, 131. Dieudonne, on cholera vibrio, 182. Digestion, relation of bacteria to, 141. Digestive fluids, action of on bacteria, 87. Dissacharides, inversion of, 131. Diseases, relation of bacteria to, 137, chap. XV. Disinfection, 84-87, 173. Dissociation, influence of on toxicity, 85, 174. Distilled water, resistance of spores to, 84. Distribution of bacteria, 44. Diphtheria, cause of, 150; bacilli of, 151, 152; growth of in milk, 117, 118; involution forms of, 27, Fig. 14 ; occurrence of Streptococcus Pyogenes in, 148 ; — toxin, immunity to, 163. Diplococcus pneumoniae, properties of, 149. Division, planes of, 18, 19. Drepanidium ranae, parasitism of, 41. Drosera, bacteria on leaf of, 138. Duclaux, on biological chemistry, 176; on milk, 176. Dulcite, fermentation of, 114; nutritive value of, 57. Dunbar, on diagnosis of cholera vibrios, 184. Dysentery, supposed cause of, 40. Dziergowski and Rekowski, on nutrition of diphtheria bacilli, 182. Ecballium elatcrinum, plasmolysis of, 5. Effront, 177; hydrofluoric method of, 120. Ehrenberg, on systematic position of bacteria, I. Khrenfest, on B. colt, 182. Ehrlich, on diphtheria serum, 184; on immunity, 183. l.idam, on resistance to desiccation, 173. /•'./(•(latins, root tubercles of, 89. Electric currents, germicidal action of, 72 ; movements induced by, 73. Elements, essential for bacteria, 54. Emmenthaler cheese, composition of, 119. Endospores, 20, Eig. 11, 21. Engelmann, bacterium method of, 61, 62, Fig. 1 6, 172; on green bacteria, 169; on purple bacteria, 68, 172. Ensilage, 120. Enzymes, 176, 178; definition of, 108; action of, no; theory of, 134. Epithelium, impenetrability of by bacteria, 142, 145. Eriksson, on breeds of Pieccinia, 93. Ernst, I So. Erysipelas, aetiology of, 148, 1 80. Erythrite, butyric fermentation of, 121. Erythrobacteria, 66 ; photosynthesis of, 68 ; rays effective in, 68, 69. Escherich, on diphtheria, 181 ; on intestinal bacteria of infants, 176, 180; on serum therapeutics, 184. Ether, action of on ferments, 109 ; lethal percentage of, 83. Ethyl alcohol, fermentation of, 112; pro- duction of by bacteria, 112, 114; by- yeast, 132. Ethylidene lactic acid, production of, 116. Eucarpous, 36. Eurythermic, definition of. 74. Exoascus, yeast of, 130. Facultative anaerobes, 61. Facultative parasites, 49. Faeces, bacteria in, 141. Fat, decomposition of by bacteria, 108 ; — during ripening of cheese, 119; occurrence of in old cultures, 14 ; per- centage of in bacteria, 52. Favus, cause of, 42. Fcchner, psycho-physical law of, So. Fehleisen, on erysipelas, 180. Fermentation, 108 ; literature of, 176, 179; biochemical explanation of, 135; theories of, 133, 134, 135; technical uses of, 123, 124, 125 ; oxidatory, 112 ; - organisms, relation of to oxygen, J35 ! ' ' products, heat of combustion of, 135, 136. Ferments, 108 ; action of, 109; organized, by-products of, no. Fermi, on non-nitrogenous bacteria, 171. Ferro-bacteria, 69. Fibrin, butyric fermentation of, 121. Finger, ( i hon and Schlagenhaufcr, on biology of Gotwcoccns, 1 8 1 . Finkler and Prior, on cholera vibrios, 182. 190 INDEX Fkcher, on cilia of bacteria, 170; on classification of, 170, 171 ; on enzymes, 176 ; on phosphorescent bacteria, 172; on plankton bacteria, 172. Fischer and Lindner, on enzymes of yeast, 178. Fission of bacteria, 16; rapidity of, 17; regular and irregular sequence of, 19. Fixing of bacteria, 6. Flagellata, relationship of to bacteria, 38. Flagellum, taxonomic importance of, 31. Flax, retting of, 123. Flexile filaments, 16. Fliigge, on decrease of virulence, 183 ; on micro-organisms, 169; -- pathogenic, 171 ; on sterilization of milk, 177. Fodder, lactic fermentation of, 1 20. Fodor, bactericidal action of blood, 183. Formic acid, production of by bacteria, 1 14. Formic aldehyde, lethal percentage of, 83. Fossil bacteria, 161 ; literature of, 182. Frank, on symbiosis, 174 ; F. and Kruger, on potato scale, 179. Frankel, on pneumonia, 181 ; germ of, 149; on nutrition of bacteria, 171. Freudenreich, on bacteria in cheese and milk, 176 ; on kephir, 177. Freund, occurrence of cellulose in tubercle bacilli, 9. Frog spawn bacterium, 124. Fruits, causes of rotting of, 99. Fumaric acid, bacterial decomposition of, 115. Fungi and bacteria, comparison between, 35, 36. Fusel oil, produ tion of, 132. Gabritschewsky, on chemotaxis of leiro- cytes, 173. Caffky, on typhoid fever, 182. Galvanotropism, 73. Ganghofner, on serum treatment of diph- theria, 184. Garden mould, presence of bacteria in, 47. Garre, on aetiology of inflammation, 180; on production of carbuncles, 148. Gartner, on heredity of tuberculosis, 181. Gases, toxicity of, 86. Gastric juice, action of on bacteria, 87, 174 ; on cholera germs, 156. Geotropism, absence of in bacteria, 73. Geppert, on disinfection, 173. Gelatine media, 57; liquefaction of, 58. Germination of spores, 20, Fig. II, 22 ; of yeast, 127. Gilbert and Uominici, on bacteria of intes- tine, 1 80. Glabrificines, 184. Glacier ice, occurrence of bacteria in, 46. Glanders germs, growth of in milk, 117, 118. Globig, on thermophile bacteria, 173. Gloedapsa, shape of, 37. Glucase, 178. Glycerine, chemotaxis of, 79 ; fermentation of, 114; nutritive value of, 57; pro- duction of by yeast, 132 ; resistance of spores to, 84 ; as source of C, 55 ; influence of on assimilation of N, 56. Glycocol, 100. Godlewski, on nitrate bacteria, 175. ' Gommose bacillaire ' of vine, 138. Gonidia, 22. Gonococci, 30, 49, 180, 143, Fig. 27 ; media for, 57 ; properties of, 148. Gonorrhoea, cause of, 148 ; -- rheumatism, cause of, 149 ; literature of, ito. Gonnermann, on root-nodule bacteria, 174. Gottstein and Schleich, 184. Gourd, min., max. and optimal temperatures for germination of, 74. Gramtlobacter, 30 ; G. lactobutyricus, 1 1 ; G. saccharobutyrtius, 121. Granulose, in bacteria, 13, 121, 139. Gregarineae, 41. Grimbert, on B, orthobutylicus, 177. Gruber, on glabrificines, 184. Gum arabic, butyric fermentation of, 121. Gumprecht, on tetanus poison, ii>2. Giinther, on Vibrio aquatilis, 182. Haemamoeba^ 40. Haemopoeic organs, 161. Haemosporidia, 41. Halibacterium, 30. Halogens, germicidal action of, 84. Hamburger, on action of gastric juice on bacteria, 174. Hansen, on acetic bacteria, 112, 176; in- fluence of heat on sporulation, 1 29 ; on Proteus, 102 ; on putrefactive bacteria, 175 ; on yeast, no, 178. Haplobacteria, 2 ; ordinal rank of, 31. Haplobacterineae, 32. Haplomycetes, 41. Havannah tobacco, bacteria of, 124. Heat, evolution of from seeds, 62, from bacteria, 63 ; influence of on ferments, 109 ; — on virulence of anthrax bacilli, 28 ; production of by blicrococctis, 63 ; resistance of spores to, 128. Heider, on Vibrio danubhtes, 182. Heim, on pathogenic bacteria of milk, butter, and cheese, 177. Helfeld, on manure bacteria, 175. Hellriegel and Wilfahrt, on assimilation of free N, 91, by Leguminoseae, 174. Hemp, retting of, 123. Herpes, 36 ; cause of, 42. Hesse, on agar culture, 172; on bacterio- logical analysis of air, 171. Hibernation of bacteria, 75. Holocarpous, 36. Homococcaceae, 33 ; chaiacters of, 32. INDEX Hoppe-Seyler, on fermentation of cellulose, 177; on oxygen and micro-organisms, '75- Human body, bacteria in, 139. Hiippe, on involution forms, 170; on lactic fermentation, 176. Hydrogen, inhibitory action of, 86 ; produc- tion of, 122. Hydrolytic action of enzymes, 109. Hydrophobia, inoculation for, 184. Hyphae, 36. Hyphomycetes, 41. Immunity, acquired, 163 ; method of pro- ducing, 163, 164; duration of, 164; hereditary, 165; literature of, 183, 184; peculiarities of, 164. Incubation period, 158. Incubators, use of, 75. Indican, fermentation of, 124. Indigo, production of, 177 ; — fermentation, 177. Indigofera iinctoria, 124. Indigoglucine, production of from indican, 124. Indol, 99, 100 ; absence of from cheese, 119; production of by cholera germs, 101, 156; by bacteria, 98 ; -- reaction, 155- Infection, 180; conditions for, 145; mode of, 144, 145 ; -- threr.d, 94, Fig. 21. Inhibitory coefficient, 82. Inoculation, principle of, 144 ; by insect stings, 145 ; methods, toxines, 163, attenuated cultures, 164 ; for small-pox, 1 68. Intestine, bacteria of, 141 ; sterility of in new-born infants, 142. Invertase, action of on cane sugar, no; properties of, 131. Involution forms, 26, 27 ; literature of, 170. Iodine trichloride, influence of on virulence of diphtheria and tetanus bacilli, 29 ; inhibitory percentage of, 82. Iwiococcus, presence of granulose in, 139. lodoform, lethal percentage of, 83. Iron, necessity of, 54; — bacteria, nutrition of, 69 ; literature of, 1 72. Isobutyl alcohol, production of by yeast, 132. Isobutyleneglycol, production of by yeast, 132. Isolation of pathogenic bacteria, methods of, 143. Janowski, influence of light on bacteria, 172. Japan, nitrite bacteria of, 105. Java, nitrate bacteria of, 105. Jenner's vaccination, 168. Kappes, on composition of bacteria, 171. Kaspareck and Kornauth, on infection of plants by anthrax, 179. Kephir, ((imposition of, 120; inversion of lactose by, 131 ; production and pro- perties of, I 19, 1 2O. Kitasato, on tetanus bacillus, 181 ; inocula- tion of, 181 ; poison of, 182. Klcchi (v.) on />'. saccharobutyricust 177; on ripening of cheese, 177. Klein, on influence of light on fungi, 172. Klocker and Schioning, on Saccliaromyces, 178. Klostcr, definition of, 32. Robert, on intoxications, 183; on ptomaines, '75- Koch, on anthrax, 2, 169, 181 ; on Bacillus^ 32 ; on B. ant bracts, 149 ; on cholera, 155, 182 ; on disinfection, 173 ; on gelatine culture, 172 ; method of, 168, 1 73 ; steamer of, 76 ; on tubercle bacilli, 152, tingibility of, 181 ; on tuberculin, 160, 182; on tuberculosis, 181. Koch and Hosaeus, on slime fungus of sugar, 178. Kornauth, on bacteria in plants, 179; on typhoid inoculation of mice, 182. Kossel, on diphtheria toxine, 182. Kossowitsch, on assimilation of N by algae, 174. Kramer, on economic importance of bacteria, 176; on mucilage production, 177; on ripening of cheese, 177. Krusse, on lysines, 184. Kuhn, on putrefactive bacteria, 175. Kiihne, on tuberculin, 182. Kurloff and Wagner, action of gastric juice on bacteria, 174. Kutscher, on phosphorescent bacteria, 172. Lactic acid, butyric fermentation of, 115, 122; occurrence of in cheese, 119; production of by bacteria, 115 ; --in rancid butter, 1 1 8 ; by Leiu onostoc, 1 24 ; in fermenting bread, 125 ; optical splitting of by bacteria, 115. Lactic bacteria, non-liquefaction of gelatine by, 58 ; in kephir and in beer, 120. Lactic fermentation, 116, 117; literature of, 176. Lafar, on acetic fermentation, 176; on acidity of yeast, 177; on spontaneous generation, 171; on technical myco- logy, 168, 176, 177. Lamella (middle)] solution of by bacteria, 123. LamprocystiS) 106 ; L. rosco-persiana, 65, Fig. 17. Lathraea, bacteria in leaf-chambers of, 138. I.averania, 40. Lead acetate, lethal percentage of, 13 ; as disinfectant, 84. Leguminosae, nitrogenous nutrition of, 89 ; partial parasitism of, 94, 95. 192 INDEX Lehmann and Neumann, 169; on Bacterium and Bacillus, 35 ; on species of bac- teria, 171 ; on fermentation of dough, 178. Leichman, on ' mucilaginous' fermentation, 177. Leipzig, nitrogen discharged by sewers of, 103. Leprosy, germ of, 153. Leptomitus, character of, 41. Leptothrix, deposition of iron in sheaths of, 69, 70 ; occurrence of in hot springs, 75; L. buccalis, 139; L. innominate, 139 ; L. ochracea, 69. Lethal coefficients, 82. Leucin, 98, 99 ; in cheese, 119 ; putrefactive decomposition of, ico ; chemotaxis of, 173' Leucocytes, ingestion of bacteria by, 161, Fig. 29. Leucocytosis, 161 ; literature of, 173. Leiiconostoc, arthrospores of, 23 ; secretion of mucilage by, 53 ; L. mesenteroides, capsulesof,io,Fig.7; formation of chains by, 19; growth of in beet-juice, 124. Leuwenhoek, discovery of bacteria by, I ; works of, 169. Lewkowitsch, on production of amygdalic acid, 176. Lichen fungi, parasitism of, 93, Fig. 20. Liesenberg and Zopf, on L. mesenteroides* 178. Life, origin of, 50; primitive character of, 51. Light, influence of on erythrobacteria and on pigment formation, 70; — on viru- lence of anthrax bacilli, 28 ; deleterious rays of. 71 ; literature of, 172 ; produc- tion of by bacteria, 63 ; influence of on spore-formation by, 72. Lindner, on technics of fermentation, 178. Lock-jaw, cause of, 150. Locomotion, organs and rapidity of in bacteria, 14. Loffler, 2; on cilia of bacteria, 170; method of staining, 14 ; on diphtheria, 8l, 82 ; on diseases of mice, 155, 182 ; on history of bacteria, 169 ; on micro- organisms of water, 171. Loffler and Abel, on typhoid immunization, 182. Lophotricha, 15; taxonomic importance of, 31- Liibbert, biology of bacteria, i£o. Luciferin, 64. Ludwig, on phosphorescent bacteria, 172. Lupinus, percentage of N in, 89 ; root- tubercle of, 90 ; L. albus, bacteroids of, 27, Fig. 14; 90, Fig. 19. Lysines, 168, 184. Magnesium carbonate, action of nitrite bacteria on, 165. Malarial fever, microbe of, 40. Maltase, properties of, 131. Mammalia, bacterial diseases of, 138, 139. • Mangin, 179. Mannite, fermentation of, 114; nutritive value of, 57 ; production of by bacteria, 123. Marine bacteria, 63, 64, 65. M assart and Bordet, on chemotaxis of leucocytes, 173. Mastigophora^ parasitism of, 39. Mayers, on fermentation, 178. Maze, 169 ; on fixation of N, 92, 174 . Melanin, occurrence of in malaria, 40. Mcngc and Kronig, on bacteria of vagina, 1 80. Mercuric chloride, inhibitory and poisonous percentages of, 82, 83. Merismopoedzci) 37. Messea, 170. Metatrophic bacteria, 54, 55 ; — fungi, 35 ; definition of, 48, 49. Methane, 100; --bacteria, 123. Methylmercaptan, 100. Methyl violet, as a disinfectant, 86. Metschnikoff, on immunity, 183 ; on phago- cytosis, 161, 183. Miasma, 142. Mice, anthrax in, 150. .Micrococcus, 33 ; M. agilis, 12 ; movement of, 14 ; M. gonorrhoeas, 143, Fig. 27 ; 1 5 1, Fig. 28; characters of, 148 ; forms of, 149. M. pyogenes, 148, 151, Fig. 28 ; M. tetragenus, 33 ; mode of division in, 19, Fig. 10 ; M.ureae, 102, Fig. 22 ; decomposition of urea by, 183. Migula, on Bacterium and Bacillus, 32; on classification of bacteria, 171. Milk, bacteria in, 117, 118; changes in- duced by, 118 ; ropiness of, 118, 123 ; butyric bacteria in, 122 ; - - sugar, in curds, 119. Miller, on bacteria of mouth, 180. Mimoseae, root- tubercles of, 91. Miquel, on bacteriological analysis of air, 171 ; on B. thermophilus, 173 ; on urea ferments, 175, 179. Molisch, on iron in plants, 172. M oiler, 172. Manas, monotrichous character of, 38. Monilia Candida, 39. Monosaccharides, fermentability of, 131. Monotricha, 15. Monotrophism, 29, 49. Morphology of bacteria, p. I seq., Chap. 1. Mosquitoes, relation of to malaria, 40. Mould fungi, fixation of N by, 97 ; organic disintegration by, 136. Mouth, bacteria of, 139, 180. Movements of bacteria, 14, 16; influence of plasmolysis on, 9 ; taxonomic value of, 31. INDEX Muoigenous bnctcria, 15. Mucilage, 53, 123 ; excretion of, 10; In l.cct- molasses, 124. Mucort parasitism of, 43 ; rotting of fruits by> 99 ! - - yeast, 1 10, 130 ; M. lOiym- bifer, Af. rlnzopoa'ifonnis, parasitism of, 43- Miiller, i. Mummies, bacteria of, 140. Miintz, on nitrification, 175. Mycelium, 36. Myco&acterium, 27; Af. tuberculosis, 153. M ycoprotein, 53. Myxomycctcs, 36. Na'geli, 2, 29; on fermentation, 179; on involution forms, i/o; on molecular movements, 169; on nutrition of bac- teria, 56, 171; on species in, 170; theory of fermentation of, 134. Neisser, on gonorrhoeal fungus, 181 ; on penetration of intestine by bacteria, I So. Nencki, on anaerobism, 172 ; on isolation of mycoprotein, 53 ; on putrefaction, 175. Xencki and Scheffer, on chemical com- position of bacteria, 171. Neoplasm, supposed origin of, 39. Nepenthes, bacteria in pitcher of, 138. Neuridine, 100. Nicolaier, on tetanus, 181. Nitragin, preparation and uses of, 93. Nitrate bacteria, 55, 105, 106. Nitrate-reducing bacteria, 106. Nitrates, as source of N, 55, 88, 94. Nitric oxide, inhibitory action of, 86. Nitrification, literature of, 175. Nitrifying bacteria, culture medium for, 54, 104, 105, Fig. 23. Nitrite bacteria, 105. Nitrites, as source of N, 54, production of, 104, 1 06. Nitrobacter, 30, 105, Fig. 23, 106. Nitrogen, 56 ; amount of discharged from sewers, 103 ; circulation of, 88, Chap. X, 98, Chap. XI ; fixation of, 96 ; liberation of, 98, 99, 104 ; influence of combination of on fermentation, 115; literature on assimilation of, 174 ; sources of, 55, 88; supposed absence of from certain organisms, 51. NitrosococcuS) 105. Nttroscmonas, 30 ; N. europea, 105, Fig. 23. N. javancnsis, 105, Fig. 23. Nitrous oxide, inhibitory action of, 86. Nobbe and Hiltner, on breeds of root- tubercle bacteria, 93. Nobbe, Hiltner, and Schmid, on root- tubercle bacteria, 174. Nocard and Roux, on tubercle bacillus, 181. No s toe, 125. Nuclei, absorption of dyes by, 7, 8. Nutrition, 52, Chap. VI ; peculiarities of, FISCHCR O 47. *]8 ; in fungi, 38 ; varj ing ( hara< UT of, 29. Nuttall, on animals and bacteria, 183. Nuttall and Thierfeldcr, on life without bacteria, 180. Oats, consumption of combined N by, 91. Obermiillcr, on tuberculous milk, 177. Obligatory aerobes, 60; — anaerobes, 61 ; - metatrophes, 49; — saprophytcs,49. Oceanic ooze, bacteria in, 64. Oedema, malignant, cause of, 150; omni- presence of germs of, 47. Oenanthic ether, production of by yeast, 32. Oidium.) 39 ; influence of on ripening of cheese, 119. Olive oil, decomposition of by bacteria, 108. Omelianski, on fermentation of cellulose, 177- Oospora, 41. Optical decompositions, 115 ; — properties, relations of to nutrition and fermenta- tion, 1 15. Oscillaria, 37 ; O. /emu's, 36, Fig. 15. Osmic acid, as fixing fluid, 6. Osmotic pressure, 5 ; in bacterial cells, 8. Oxygen, influence of on alcoholic fermenta- tion, 132, 178 ; --on growth of yeast, 133] — on ciliary movement in aerobes and anaerobes, 61 ; — on pigment formation, 61 ; — on putrefaction, 100 ; — on spore formation, 150 ; relation of to fermentative organisms, 135. Ozone, inhibitory action of, 86. Pancreatic juice, action of on bacteria, 87. Papilionaceae, root-tubercles of, 91. Paracasein, occurrence of in curds, 119. Parachromatophores, 13. Paraoxyphenylacetic acid, 100. Parasites, 47 ; bacterial, 144. Parasitism, influence of temperature on, 43. Paratrophic, 48, 49 ; — bacteria, 57 ; - fungi, 36. Passet, on aetiology of Pneumonia, 180. Pasteur, 2; on anaerobiosis, 60, 133, 172 ; dictum of, 134 ; on fermentation, no; influence of heat on anthrax, 29; on organized ferments, 108 ; on production of racemic acid, 176; on protective inoculation for rabies, 168, 184 ; -- for anthrax, 168 ; on silkworm disease, 138, 1 80 ; on spontaneous generation, 171; studies on beer, 179; wine, 177; works of, 176. Pasteur, Chamberland, and Roux, on attenuation, 170. P. and Joubcrt, on fermentation of urine, 175. Pasteurixation of wine, 75. Pathogenic bacteria, 151, Fig. 28 ; isolation of, 143; culture and attenuation of, 144; mode of action of, 158, 159 ; products 194 INDEX of, 159, 160 ; relation of to host, 146 ; varieties and races of, 147. Paul and Kronig, action of chemicals on bacteria, 173. Pea, butyric fermentation of, 121 ; fixation of N by, 91. Pectin compounds, action of retting bacteria on, 123. Pediococcus, 33 ; P. tetragenus, 19, Fig. 10. Pellicles, formation of, 3 ; character of, 4. Penicillium, growth of on fruits, 99 ; P. glaticum, 3, Fig. 2 ; 36, Fig. 15 ; influence of on ripening of cheese, 119. Pentamethylendiamine, loo. Pepsin, action of on proteids, no. Peptone, chemotaxis of, 79 ; minimal amount for, 8 1 ; properties of, 99 ; as source of N and of C, 55 ; - - bacteria, 55- Pe're, on lactic fermentation, 176. Perithecia, 42. Peritricha, 15 ; taxonomic importance of, 31. Permeability of bacteria, 8, 9. Peters, on fermentation of dough, 1/8. Petruschky, on pathogenic Streptococci, I So. Pettenkofer, on cholera, 182. Pettenkofer and Emmerich, on cholera germs, 155. Pfeffer, on chemotaxis, 79, 173; minimal amount of peptone for, Si ; on Weber's Law, 173 ; on symbiosis of Lichens, 95. Pfeiffer, cholera serum reaction of, 166; on diagnosis of cholera Vibrio, 184; on immunity, 155. Pfeiffer and Proskauer, on cholera antitoxin, 184. Pfliiger, on phosphorescent bacteria, 172. Phagocytes, ingestion of bacteria by, 161 ; chemotropism of, 162. Phagocytosis, literature of, 182, 183. Phenol, 99 ; action of on ferments, 109 ; on virulence of bacteria, 28. Phenylacetic acid, 99. Phenylamido-propionic acid in cheese, 119. Phenylpropionic acid, 99. Phisalix, on asporogenous anthrax, 170. Phosphorescence, of bacteria, 63, 172 ; influence of oxygen on, 63 ; culture of, 63, 64. Phosphuretted hydrogen, production of, loo. Photobacterium, 30, 63. Phototactic sensitiveness, 69. Phthisis, occurrence of Streptococcus pyo- genes in, 148. Pigments, of bacteria, 12 seq. ; in milk, 118; distribution of, 12, 13; influence of medium on, 56 ; of oxygen on, 61 ; in yeasts, 128. Pilobolus, influence of light on formation of sporangia of, 72. Pinguicula, bacteria on leaf of, 138. Planococcus, 33. Planosarcina, 33. Plasmodium malariae, 40. Plasmolysis, 6 ; of bacteria, 8, Fig. 6 ; influence of on movement, 9. Plate culture, 58. Plectridiae, 33. riectridhim, 33 ; types of, 121 ; P.paludo- sum, in, Fig. 24 ; formation of spores of, 20, Fig. ii. P. tetani, 150. Plectron, 32 ; of anthrax, 31. Pleogeny, 24, 29. Pleomorphism, 274; literature of, 170; in haplo-bacteria, 25. Pleurococcus, 37. PneumococcHS, 149 ; fermentative activity of, 114. Pneumonia, cause of, 149. Poikilothermism of bacteria, 73. Poisons, chemotaxis of, 79, So ; modification of action of, 81, 82 ; lethal and inhi- bitory percentages of, 82, 83. Polar granules, 9. Pollender, on anthrax in blood, 181. Polysaccharides, non-fermentability of by yeast, 131. Polytoma urella, 38. Polytrophic bacteria, 29. Polytrophism, 49. Popoff, on fermentation of dough, 178. Potash, caustic, lethal percentage of, 83,84. Potassium salts, chemotaxis of, 79 ; nitrate as source of N, 55 ; - - perman- ganate, lethal percentage of, 83, 84. Potato, bacterial diseases of, 138. Prazmowski, on development of bacteria, 170; on involution forms, 170; on root-tubercle bacteria, 174. Predisposition, 167. Pressure, influence of on bacteria, 73, 173. Primitive organisms, theories of, 7. Propionic acid, production of by bacteria, 114. Propyl alcohol, production of by yeast, 132. Proskauer and Beck, on tuberclebacillus, 1 8 1 . Proteids, percentage of in bacteria, 52 ; decomposition of, 99, 100. Proteus, 30, 102; P. vulg art's, 101, 102, Fig. 22. Protista, 35 ; place of in nature, 36. Prototrophic bacteria, 54. Prototrophism, 48, 49. Protozoa, importance of in putrefaction, 136. Pseudomonas, 32. Pseudopodia, 40. Psichohormiuin, iron in, 70. Psycho-physical law, So. Ptomaines, importance of, 159; literature of, 175 ; production of, 100. Ptomatropine, loo. Puccinia, cultural varieties of, 93. Pulmonary phthisis, origin of, 152. Purple bacteria, 172. INDEX 195 Pus-luu'tena, 148, 149; - - cocci, results of injection of, 145. Putrefaction, 101 ; influence of oxygen and proteids on, loo ; of temperature and water on, 99 ; peculiarities of, 102, 103 ; liberation of N by, 104 ; ultimate pro- ducts of, 101 ; - - in intestine, 141. Putrefactive bacteria, 102, Fig. 22 ; litera- ture of, 175 ; zymogenic properties of, 102. Putrescine, 100. Pyaemia, 145 ; causes of, 148. Pykotanine, as disinfectant, 86. Pyogenic diseases, 148. Pyrenomycetes, disintegration of wood by, 136- Quarter-evil of cattle, causes of, 120, 150. Quinine, inhibitory percentage of, 82. Rabies, inoculation for, 168 ; Pasteur's treatment of, 184. Rabinowitsch, on thermophile bacteria, 173. Racemic acid, optical decomposition of by bacteria, 115. Radicles, basic and acid, chemotaxis of, 79. Ragi, symbiotic character of, no. Raphidia, 37. Rathay, on gum-producing bacteria, 1 79. Ray-fungus, parasitism of, 41 ; sporangia of, 42. Rayer, on anthrax bacilli m blood, 181. Red sulphur bacteria, nutrition of, 68. Rees, on yeasts, 178. Renault, on fossil bacteria, 183. Rennet, production of by bacteria, 117, 1 18. Reproduction, of bacteria, by fission, 16 seq. ; rapidity of, 17 ; by spore forma- tion, 19 seq. Respiration, of bacteria, 60 ; intramolecular, 133. Retting bacteria, 123 ; of flax, 177. Rhinanthaceae, partial parasitism of, 96. Rhizobium Icgiuninosaruni, 92. Riedler, on leucocytosis, 173. Ring-worm, cause of, 42. Rontgen rays, action of on bacteria, 73. Root-nodules, 89 ; intercellular spaces in, 174; literature of, 174; percentage of N in, 95; structure of, 90, Fig. 19; bacteria of, assimilation of N by, 89, 92 ; - shape and properties of, 90, Fig. 19, 92, 93 ; artificial cultivation of, 92 ; penetration of, 94 ; growth and nutrition of, 95, Fig. 21. Roquefort cheese, ripening of, 119. Rosenbach, on infection of wounds, 180. Rothenbach, on use of pure yeast, 177. Roux, on antitoxic serums, 183, i£'4; on asporogenous anthrax, 170; on bac- teriological analysis of water, 171. Roux and Martin, on diphtheria, 183. Roux and Ycrsin, on diphtheria, iSl ; preparation of toxin of, 182. Rubidium, chemotaxis of, 79; substitution of for potassium, 54. Russel, on nitrate bacteria of oceanic oo^e, 65, SaccharomyceSy determination of species in, 128; forms of, 126, 127, Fig. 25; nutrition of, 131 ; pathological proper- ties of, 36 ; symbiosis of in kephir, 1 20; systematic position of, 130; S. albicans, parasitism of, 39 ; S. cere- vt'siae, races of, in ; sporulation of, 128; S. cllipsoidcus, ill ; sporulation of, 128 ; S.g/utinis, 128 ; S. mycoderma, decomposition of alcohol by, 112 ; S, PasteurianuS) sporulation of, 128. Sake", preparation of, 1 10. Salicylate of sodium, inhibitory percentage of, 82. Salicylic acid, lethal percentage of, 83 ; action of on ferments, 109. Saliva, action of on bacteria, 87. Saltpetre beds, 104. Salt solution, influence of on toxicity of sublimate, 86. Sanarelli, on water bacteria, 1 80. Saprogenic bacteria, 29 ; putrefactive power of, 101. Saprophile bacteria, definition of, 49 ; pro- perties of, 101. Saprophytes, 47. Sarcina, 33; mode of division in, 19, Fig. 10; social aggregates of, 31 ; S. ventriculi, 141. Sarcosporidia, 41. Sauerkraut, production of, 121. Scherffel, on leaf scales of Latkraea, 1 80. Schild, on intestinal bacteria of infants, 1 80. Schizomycetes, definition of, 35. Schizophyta, definition of, 37. Schloesing and Laurent, on fixation of N by bacteria, 174. Schlossberger, on mycoprotein in yeast, 53. Schorf of potato, 138. Schreiber, spore formation in bacteria, 170. Schroeter, on bacterial pigments, 169. Sderothrix Kochii, 153. Scytonemeae, relationship of to Cladothrix^ 1 *7 Sea-water, percentage of bacteria in, 64. Selective power, 115. Selmi, on ptomaines, 175. Septicaemia, 145; causes of, 148. Sero-therapeutics, investigations in, 161 ; principles of, 144 ; literature of, 184. Serum, bactericidal influence of, 162 ; cura- tive, 165 ; — for cholera, 167 ; — mode of production of, 164; — treatment, literature of, 183, 184. O 2 196 INDEX Sheaths, occurrence of, 10 ; ferruginous modification of, 11. Silkworm, disease of, 138. Silver nitrate, inhibitory percentage of, 82 ; lethal, 83. Sinapis alba, non-fixation of N by, 89. Skatol, absence of from cheese, 119; pro- duction of, 98 ; properties of, 99. Small-pox, vaccination for, 168. Soda, caustic, inhibitory percentage of, 82 ; inefficiency of as disinfectant, 84. Sodium, chemotaxis of, 79 ; necessity of, 54 ; — chloride, inhibitory percentage of, 82 ; — resistance of spores to, 84. Sommaruga, on metabolism of yeast, 176. South America, nitrite bacteria of, 105. Soy, preparation of, no. Specific characters, of bacteria, 24, 29 ; variations of due to environment, 25-28. Spirillaceae, 34. Spirillum, 1,34; sap vacuoles of, 6 ; shape of, 2 ; S. desulphuricans, reduction of sulphates by, 106; S. rnbntin, 12; influence of O on pigmentation of, 61 ; 6". split igenurn, I, Fig. I ; S. undula, 3, Fig. 2 ; 7, Fig. 5 ; cilia of, 15, Fig. 8 ; plasmolysis of, 8, Fig. 6 ; polar granules of, 9; saprophile character of, 101. Spirochaetet 2, 34 ; S. dentinm, 139, 140, Fig. 26 ; S. Obermaieri, 3, Fig. 2. Spirit Una, shape of, 37. Spontaneous generation, 50 ; literature of, 171 ; — combustion, causes of, 63. Spores, of bacteria, 19 seq. ; shapes of, 20, 21, Fig. 1 1 ; germination of, 20, Fig. 1 1, 22 ; influence of heat on, 128 ; influence of desiccation on, 76 ; non-penetration of plants by, 137 ; resistance of to poisons, 83, 84 ; staining of, 21 ; litera- ture of, 170; ubiquitous distribution of, 50 ; vitality of, 22 ; of Saccharomyces, 127, Fig. 25. Spore formation, causes of, 23 ; influence of heat on, 129, 150; of oxygen on, 150. Spore membrane, influence on vitality of spore, 76. Sporozoa, parasitism of, 41. Sporulation, taxonomic value of, 31. Spree, bacteria of, 46. Sputum, influence of on action of poisons, S3- Stab-culture, 58 ; diagnostic value of, 59. Stahl, on trophotropism, 78, 173. Staining of bacteria, 6, 7. Staphylococcus, size of, 4 ; immutability of, 25; mode of division of, 19; S.pyogenes albtts, 148; S.pyogenes aurcus, 151, Fig. 28; characters of, 148 ; colour of, 12 ; S.pyogenes titreus, 148. Starch, fermentation of, 121. Stenothermic, 74. Stereo-isomers, production of by bacteria, A1*'. Sterilization, 51. Stichococcus, 37. Stomach, bacteria of. 140 ; influence of acidity of on, 141. Streak-culture, 59. Streptococcus, 33 ; growth of in fluid media, 58 ; non-liquefaction of gelatine by, 58 ; S. pyogenes, 151, Fig. 28; characters of, 148 ; chains of, 19, Fig. 10. Streptotkrix, taxonomic position of, 41 ; S.actinoinyces,\\ ; supposed sporangia of, 42. Stutzer and Hartleb, on nitrifying fungi, 175. Sublimate, inhibitory percentage of, 82 ; lethal, 83. Succinic acid, loo; nutritive value of, 57; production of by bacteria, 114; — in kephir, 120; — by yeast, 132. Sugar, butyric fermentation of, 121 ; chemo- tactL attraction of, 79 ; influence of on fixation of N ; nutritive value of, 57 ; - as source of C, 55. Sulphur, in bacteria, 14, 65, 66. Sulphur-bacteria, 65, Fig. 17; literature of, 172; nutrition of, 66, 67; respiration of, 68; saprophile character of, 101. Sulphur dioxide, inhibitory action of, 86. Sulphuretted hydrogen, inhibitory action of, 86. Sulphuric acid, lethal percentage of, 83 ; inefficiency of as disinfectant, 84. Sunlight, germicidal action of, 71. Suppuration, character and causes of, 147, 148. Sycosis, causes of, 148. Symbiosis, 93 ; of bacteria and carnivorous plants, 35. Tartaric acid, nutritive value of, 57. Taxonomy, 24, Chap. III. Teeth, bacterial decay of, 140. Temperature, influence of an alcoholic fermentation, 132 ; on cholera germs, 156 ; on growth of Leuconostoc, 124 ; - on parasitism, 43 ; — on production of poison, 1 57; — of yeast spores, 128; — of bacterial spores, 129, 150 ; — on toxicity, 86 ; on urase, 179; resistance of bac- teria to changes of, 19 ; literature of, 173 ; high, germicidal action of, 75 ; resistance of spores to, 75 ; influence of on toxines, 160; on putrefaction, 99; low, resistance to, 75 ; maxima, dia- gnostic value of, 1 12 ; optimum for acetic fermentation, 113 ; relation of bacteria to, 73-75- Tertiary epoch, bacteria of, 161. Tetanus, aetiology of, 181 ; artificial immu- nity to, 164; cause of, 150; influence of antitoxins on, 165 ; poison of, 182 ; L\l)E.\ 197 - bacilli, presence of in soil, 147; - toxinc, 1 60. Tctramitus, ciliation of, 38. Thermogenic bacteria, 63. Thermogenous bacteria, 172. Thermophilic bacteria, 74 ; literature of, 173. Tticsiieni, partial parasitism of, 96. Thiobactcria, 65. Thit'cystis, pigment in, 12. Thiopcdia, 66 ; division of, 19; systematic position of, 33. Thiospirillum^ 66. 'J'hiotliri.v, 34, 66; arthrospores of, 22. Thrush, origin of, 39. Thymol, inhibitory percentage of, 82 ; lethal, 83. Tischutkin, on bacterial digestion, 180. Tissues, pathogenic bacteria of, 145 ; de- monstration of in, 142, 143. Tobacco, fermentation of, 124. Tolypothrix, 37. Toxalbumines, 53, 160. Toxic and antitoxic units, 166. Toxicity, influence of mixed solutions on, 86; relationship of dissociation to, 85. Toxines, intracellular, 160 ; preparation and properties of, 159 ; poisonous character of, 160; toleration of, 165; literature of, 182-184. Trcuiescantia, cell-division in, 18. Traube, theory of fermentation, 134. Traumatic infection, 150. Trichobacteria, definition of, 2 ; ordinal rank of, 31. Trichobacteriaceae, 34. Trichomonas intestinalis, 39 ; T. vaginalis, 39- Trichophyton totisurans, parasitism of, 42. Trimethylamine, 100. Trophotropism, 78, 173. Tubercle, cause of, 152; germs of, 153; — bacilli, growth of in milk, 117; invo- lution forms of, 170 ; lethal percentages of different poisons for, 81,83; sources of, 118; in sputum, 143, Fig. 27; steno- thermic character of, 74 ; vitality of, 144. Tuberculin, composition and preparation of, 160; literature of, 182. TuberculomyceS) 27, 153. Tuberculosis, aetiology of, iSl ; influence of on milk, 118; transference of, 152. Turgor, definition of, 5. Typhoid bacilli, 7, Fig. 5 ; decomposition of fat by, 108 ; growth of in milk, 117, 118; plasmolysis of, 8, Fig. 6; poly- trophism of, 29 ; suppuration due to, 147 ; — fever, cause of, 154 ; aetiology of, i Si. Tyrosin, 99 ; occurrence of in cheese, 119 ; production of by bacteria, 98 ; putre- faction of, loo. Tyiothri.-c, in cheese, 119; curdling of milk by, 118. Tyrotoxine, 100. Urase, 178 ; mode of action of, 135. Urea, fermentation of, 134; nutritive value of, 57 ; — bacteria, 103 ; literature of, 175; Urobacillus Sckutaenbergii, 179. Ustiliigti, yeast of, 1 30 ; U. carbo, resistance of spores of to desiccation, 79. Vaccine lymph, 168 ; — microbe, 40. Vacuoles in bacteria, 6 ; demonstration of, 7, Fig. 5. Vaill.ird, on hereditary immunity, 183. Valerianic acid, 100. Valeric acid, 100. Van Laer, on alcoholic fermentation, 177. Van Tieghem, on li.ic. amyloboctcr, 177; on fossil butyric bacteria, 182; on Leuconostoc mesenteroides, 178. Vegetables, mucilaginous fermentation of, 123 ; souring of, 120. Verworn, on Protista, 172. Vibrio, I, 102, Fig. 22; characters of, 34; shape of, 2 ; V. albansis, phospho- rescence of, 63 ; V. berolincnsis, patho- genicity of, 156; V. buccalts, I, Fig. I ; 139, 140, Fig. 26 ; V. cholerae, 3, Fig. 2 ; 7, Fig. 5; 151, Fig. 28; cilia of; 15, Fig. 8 ; culture of, 55, 156 ; plasmolysis of, 8, Fig. 6 ; polar granules of, 9 ; sensitiveness of to acid, 56 ; V. danu- bicus, pathogenicity of, 156; V. rugula, production of methane by, 121. Vibrion butyrique of Pasteur, 121. Vicia villosa, bacteroids of, 27, Fig. 14 ; 90, Fig. 19. Vine, bacterial diseases of, 138. Vinegar, manufacture and fermentation of, no, in, 113. Virulence, definition of, 160. Virus animatiim, 142 ; — inanimum, 159. Vital air, 60. Voges, on immunity to cholera, 182. Warington, on nitrification, 175. Water, percentages of in plants and ani- mals, 52; presence of bacteria in, 45 ; bacteriological analysis of, 46. Watson- Cheyne, on infection, 180; on Bac. anthracis, 179. Watson-Cheyne and Cheshire, on Bacillus alvei, 180. Weber's Law, 80. Wehmer, on Aspergillus yeast, 176; on decay of fruits, 175. Weichselbaum, on inflammation of lungs, 181. Weizmann, on ripening of cheese, 177. Welter, influence of cold on bacteria, 173. 198 INDEX Went and Prinsen Geerligs, on Aspcrgillus yeast, 176. Wertheim, on gonorrhoea, 181. Wheat, minimum, optimum, and maximum temperatures for germination of, 74 ; percentage of nitrogen in, 74. Wine, cause of sweetness of, 132 ; influence of bacteria on acidity of, 114 ; lactic fer- mentationof, 120; mucilaginousfermen- tation of, 123 ; — yeasts, races of, 129. Winogradsky, on fixation of N by soil bacteria, 96, 174; on iron bacteria, 69, 142; nitrifying bacteria, 104, 175;- plate-culture of, 105 ; retting bacteria, 177 ; sulphur bacteria, 65, 172. Wittlin, 173. Wladimiroff, on antitoxin and tetanus poison, 184. Wolffhiigel, on bacteriological analysis of water, 171. Wollny, on humus, 175. Woronin, on bacteroids of root-nodules, 90; on root-nodules, 174. Wortmann, on pure yeasts, 178. Wounds, aseptic treatment of, 87 ; healing of in plants and animals, 145 ; non- dangerous character of in plants, 138. Xanthoria parictina^ 93, Fig. 20. Yeasts, 126 seq. ; colonies and spores of, 127, Fig. 25 ; sterilization of, 120; in- fluence of heat on sporulation of, 129 ; literature of, 178; wild varieties of, 131 ; variations in, 178. Yersin, on antiseptics, 173. Zoogloea, formation of, 3 ; characters of, 4. Zoogloca rainigcra, 4, Fig. 3. Zopf, on life cycles of bacteria, 29 ; on pleomorphism in bacteria, 170. Zymase, 179. THE END OXFORD I PRINTED AT THE CLARENDON PRESS BY HORACE HART, M.A., PRINTER TO THE UNIVERSITY •IB 1 HnB