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Bacteriology

form

the zoogloea is now known to be a sort of resting condition of the Schizomycetes, the various elements being glued together, as it were, by their enormously swollen and diffluent cell-walls becoming contiguous. The zoogloea is formed by active division of single or of several mother-cells, and the progeny appear to go on secreting the cell-wall substance, which then absorbs many times its volume of water, and remains as a consistent matrix, in which the cells come to rest. The matrix - i.e. the swollen cell-walls - in some cases consists mainly of cellulose, in others chiefly of a proteid substance; the matrix in some cases is horny and resistant, in others more like a thick solution of gum. It is intelligible from the mode of formation that foreign bodies may become entangled in the gelatinous matrix, and compound zoogloeae may arise by the apposition of several distinct forms, a common event in macerating troughs (fig. 3, A). Characteristic forms may be assumed by the young zoogloea of different species, - spherical, ovoid, reticular, filamentous, fruiticose, lamellar, &c., - but these vary considerably as the mass increases or comes in contact with others. Older A FIG. 7. - Bacillus e nthracis. (After Koch.) A. Bacilli mingled with blood-corpuscles from the blood of a guinea-pig; some of the bacilli dividing.

humour. They grow out into long leptothrix-like filaments, which B. The rodlets after three hours' culture in a drop of aqueous become septate later, and spores are developed in the segments. (X 650) .

zoogloeae may precipitate oxide of iron in the matrix, if that metal exists in small quantities in the medium. Under favourable conditions the elements in the zoogloea again become active, and move out of the matrix, distribute themselves in the surrounding medium, to grow and multiply as before. If the zoogloea is formed on a solid substratum it may become firm and horny; immersion in water softens it as described above.

The growth of an ordinary bacterium consists in uniform elongation of the rodlet until its length is doubled, followed by division by a median septum, then by the simul- Measure- taneous doubling in length of each daughter cell, again ment of followed by the median division, and soon (figs. 13, 14). growth. If the cells remain connected the resulting filament repeats these processes of elongation and subsequent division uniformly so long as the conditions are maintained, and very accurate measurements have been obtained on such a form, e.g. B. ramosus. If a rodlet in a hanging drop of. nutri' nt gelatine is fixed under the microscope and kept at constant temperature, a curve of growth can be obtained recording the behaviour during many hours or days. The measured lengths are marked off on ordinates erected on an abscissa, along which the times are noted. The curve obtained on joining the former points then brings out a number of facts, foremost among which are (1) that as long as the conditions remain constant the doubling periods - i.e. the times taken by any portion of the filament to double its length - are constant, because each cell is equally active along the whole length; (2) there are optimum, minimum and maximum temperatures, other conditions remaining constant, at which growth begins, runs at its best and is soon exhausted, respectively; (3) that the most rapid cell-division and maximum growth do not necessarily accord with the best conditions for the life of the organism; and (4) that any sudden alteration of temperature brings about a check, though a slow rise may accelerate growth (fig. 8). It was also shown that exposure to light, dilution or exhaustion of the food-media, the presence of traces of poisons or metabolic products check growth or even bring it to a standstill; and the death or injury of any single cell in the filamentous series shows its effect on the curve by lengthening the doubling period, because its potential progeny have been put out of play. Hardy has shown that such a destruction of part of the filament may be effected by the attacks of another organism.

80 85 1 J FIG. 8. - Curve of growth of a filament of Bacillus ramosus (Fraenkel), constructed from data such as in fig. 4. The abscissae represent intervals of time, the ordinates the measured lengths of the growing filament. Thus, at 2.33 the length of the filament was 6; at 5.45, at 8 P.M., 70 and so on. Such curves show differences of steepness according to the temperature (see temp. curve), and to alterations of light (lamp) and darkness. (H. M. W.) A very characteristic method of reproduction is that of sporeformation, and these minute reproductive bodies, which represent a resting stage of the organism, are now known in many spores. forms. Formerly two kinds of spores were described, arthrospores and endospores. An arthrospore, however, is not a true spore but merely an ordinary vegetative cell which separates and passes into a condition of rest, and such may occur in forms which form endospores, e.g. B. subtilis, as well as in species not known to form endospores. The true spore or endospore begins with the appearance of a minute granule in the protoplasm of a vegetative cell; this granule enlarges and in a few hours has taken to itself all the protoplasm, secreted a thin but very resistive envelope, and is a ripe ovoid spore, smaller than the mother-cell and lying loosely in it (cf. figs. 6, 9, io, and II). In the case of the simplest and most minute Schizomycetes III. 6 280 260 240 220 200 180 160 140 120 100 80 60 40 20 2pm, 2.33 S.3S 3.5 3.30 S.9S 620 7.7 (Micrococcus, &c.) no definite spores have been discovered; any one of the vegetative micrococci may commence a new series of cell by growth and division. We may call these forms " asporous," at any rate provisionally.

The spore may be formed in short or long segments, the cellwall of which may undergo change of form to accommodate itself to the contents. As a rule only one spore is formed in a cell, and the process usually takes place in a bacillar segment. In some cases the spore-forming protoplasm gives a blue reaction with iodine solutions. The spores may be developed in cells which are actively swarming, the movements not being interfered with by the process (fig. 4, D). The so-called " KOpfchenbacterien " of older writers are simply bacterioid segments with a spore at one end, the mother cell-wall having j adapted itself to the outline of the spore 2 (fig. 4, F). The ripe spores of Schizomy y 0 cetes are spherical, ovoid or long-ovoid in ab shape and extremely minute (e.g. those of Bacillus subtilis measure o. 0012 mm. long by o.0006 mm. broad according to Zopf), highly refractive and colourless (or very dark, probably owing to the high index of refraction and minute size). The membrane may be relatively thick, and even exhibit shells or strata.

The germination of the spores has now been observed in several forms with care.

A The spores are capable of germination at once, or they may be kept for months and even years, and are very resistant against desiccation, heat and cold, &c. In a suitable medium and at a proper temperature the germination is completed in a few hours. The spore swells and elongates and the contents grow forth to a cell like that which produced it, in some cases clearly breaking through the membrane, the remains of which may be seen attached to the young germinal rodlet (figs. 5, 9 and II); in other cases the surrounding membrane of the spore swells and dissolves. The germinal cell then grows forth into the forms typical for the particular Schizomycete concerned.

The conditions for spore-formation differ. Anaerobic species usually require little oxygen, but aerobic species a free supply. Each species has an optimum temperature and many are known to require very special food-media. The systematic interference with these conditions has enabled bacteriologists to induce the development of socalled asporogenous races, in which the formation of spores is indefinitely postponed, changes in vigour, virulence and other properties being also involved, in some cases at any rate. The addition of minute traces of acids, poisons, &c., leads to this change in some forms; high temperature has also been used successfully.

The difficult subject of the classification of bacteria dates ' The difficulties presented by such minute and simple organisms as the Schizomycetes are due partly to the few " characters " which they possess and partly to the dangers of error in manipulating them; it is anything but an easy matter either to trace the whole development of a single form or to recognize with certainty any one stage in the development unless the others are known. This being the case, and having regard to the minuteness and ubiquity of these organisms, we should be very careful in accepting evidence as to the continuity or otherwise of any two forms which falls short of direct and uninterrupted observation. The outcome of all these considerations is that, while recognizing that the " genera " and " species " as defined by Cohn must be recast, we are not warranted in uniting any forms the continuity of which has not been directly from the year 1872, when Cohn published his system, which was extended in 1875; this scheme has in fact dominated the study of bacteria ever since. Zopf in 1885 proposed a scheme based on the acceptance of extreme views of pleomorphism; his system, however, was extraordinarily A FIG. I o. - Bacillus subtilis. (After Strasburger.) A. Zoogloea pellicle (X 500). B. Motile rodlets (X 1000). C. Development of spores (X 800).

impracticable and was recognized by him as provisional only. Systems have also been brought forward based on the formation of arthrospores and endospores, but as explained above this is eminently unsatisfactory, as arthrospores are not true spores and both kinds of reproductive bodies are found in one and the same form. Numerous attempts have been made to construct schemes of classification based on the power of growing colonies _ ? J ?

P.M. - -.__-- ,:?

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?;?. ????? ?4 ?. -- FIG. I I. - Stages in the development of spores of Bacillus ramosus (Fraenkel), in the order and at the times given, in a hanging drop culture, under a very high power. The process begins with the formation of brilliant granules (A, B); these increase, and the brilliant substance gradually balls together (C) and forms the spores (D), one in each segment, which soon acquire a membrane and ripen (E). (H. M. W.) to liquefy gelatine, to secrete coloured pigments, to ferment certain media with evolution of carbon dioxide or other gases, or to induce pathological conditions in animals. None of these systems, which are chiefly due to the medical bacteriologists, has maintained its position, owing to the difficulty of applying the characters and to the fact that such properties are physiological and liable to great fluctuations in culture, because a given organism may vary greatly in such respects according to its degree of vitality at the time, its age, the mode of nutrition observed; or, at any rate, the strictest rules should be followed in accepting the evidence adduced to render the union of any forms probable.

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FIG. 9.

A, Bacillus anthracis. (After de Bary.) Two of the long filaments (B, fig. io), in which spores are being developed. The specimen was cultivated in broth, and spores are drawn a little too small - they should be of the same diameter transversely as the segments. (X 600.) B. Bacillus subtilis. (After de Bary.) "1, fragments of filaments with ripe spores; 2-5, successive stages in the germination of the spores, the remains of the spore attached to the germinal rodlets. (X 600.) P.M. and the influence of external factors on its growth. Even when used in conjunction with purely morphological characters, these physiological properties are too variable to aid us in the discrimination of species and genera, and are apt to break down at critical periods. Among the more characteristic of these schemes adopted at various times may be mentioned those of Miguel (1891), Eisenberg (1891), and Lehmann and Neumann (1897). Although much progress has been made in determining the value and constancy of morphological characters, we are still in need of a sufficiently comprehensive and easily applied scheme of classification, partly owing to the existence in the literature of imperfectly described forms, the life-history of which is not yet known, or the microscopic characters of which have not been examined with sufficient accuracy and thoroughness. The principal attempts at morphological classifications recently brought forward are those of de Toni and Trevisan (1889), Fischer (1897) and Migula (1897). Of these systems, which alone are available in any practical scheme of classification, the two most important and most modern are those of Fischer and Migula. The extended investigations of the former on the number and distribution of cilia (see fig. I) led him to propose a scheme of classification based on these and other morphological characters, and differing essentially from any preceding one. This scheme may be tabulated as follows: I. O [[Rder - I]]-laplobacterinae. Vegetative body unicellular; spheroidal, cylindrical or spirally twisted; isolated or connected in filamentous or other growth series.

I. Family - CoecACEAE. Vegetative cells spheroidal.

(a) Sub-family - ALLOcoccAcEAE. Division in all or any planes, colonies indefinite in shape and size, of cells in short chains, irregular clumps, pairs or isolated :- Micrococcus (Cohn), cells non-motile; Planococcus (Migula), cells motile.

(b) Sub-family - HoMococcACEAE. Division planes regular and definite : - Sarcina (Goods.), cells non-motile; growth and division in three successive planes at right angles, resulting in packet-like groups; Planosarcina (Migula), as before, but motile; Pediococcus (Lindner), division planes at right angles in two successive planes, and cells in tablets of four or more; Streptococcus (Billr.), divisions in one plane only, resulting in chains of cells.

2. Family - Bacillaceae. Vegetative cells cylindric (rodlets), ellipsoid or ovoid, and straight. Division planes always perpendicular to the long axis.

(a) Sub-family - Bacilleae. Sporogenous rodlets cylindric, not altered in shape : - Bacillus (Cohn), non-motile; Bactrinium (Fischer), motile, with one polar flagellum (monotrichous); Bactrillum (Fischer), motile, with a terminal tuft of cilia (lophotrichous); Bactridium (Fischer), motile, with cilia all over the surface (peritrichous) .

Sub-family - Clostridieae. Sporogenous rodlets, spindle-shaped : - Clostridium (Prazm.), motile (peritrichous).

(c) Sub-family - Plectridieae. Sporogenous rodlets, drumstick-shaped : - Plectridium (Fischer), motile (peritrichous).

3. Family - SPIRILLAcEAE. Vegetative cells, cylindric but curved more or less spirally. Divisions perpendicular to the long axis: - Vibrio (Muller-Ldffler), commashaped, motile, monotrichous; Spirillum (Ehrenb.), more strongly curved in open spirals, motile, lopho trichous; Spirochaete (Ehrenb.), spirally coiled in numerous close turns, motile, but apparently owing to flexile movements, as no cilia are found.

II. O Rder - Trichobacterinae. Vegetative body of branched or unbranched cell-filaments, the segments of which separate as swarm-cells (Gonidia). I. Family - Trichobacteriaceae. Characters those of the Order.

(a) Filaments rigid, non-motile, sheathed: - Crenothrix (Cohn), filaments unbranched and devoid of sulphur particles; Thiothrix (Winogr.), as before, but with sulphur particles; Cladothrix (Cohn), filaments branched in a pseudo-dichatomous manner.

(b) Filaments showing slow pendulous and creeping movements, and with no distinct sheath: - Beggiatoa (Tre y .), with sulphur particles.

The principal objections to this system are the following: - (1) The extraordinary difficulty in obtaining satisfactory preparations showing the cilia, and the discovery that these motile organs are not formed on all substrata, or are only developed during short periods of activity while the organism is young and vigorous, render this character almost nugatory. For instance, B. megatherium and B. subtilis pass in a few hours after commencement of growth from a motile stage with peritrichous cilia, into one of filamentous growth preceded by casting of the cilia. (2) By far the majority of the described species (over woo) fall into the three genera - Micro- coccus (about 400), Bacillus (about 200) and Bactridium (about 150), so that only a quarter or so of the forms are selected out by the other genera. (3) The monotrichous and lophotrichous conditions are by no means constant even in the motile stage; thus Pseudomonas rosea (Mig.) may have I, 2 or 3 cilia at either end, and would be distributed by Fischer's classification between Bactrinium and Bactrillum, according to which state was observed. In Migula's scheme the attempt is made to avoid some of these difficulties, but others are introduced by his otherwise clever devices for dealing with these puzzling little organisms.

The question, What is an individual ? has given rise to much difficulty, and around it many of the speculations regarding pleomorphism have centred without useful result. If a tree fall apart into its constituent cells periodically we should have the same difficulty on a larger and more complex scale. The fact that every bacterial cell in a species in most cases appears equally capable of performing all the physiological functions of the species has led most authorities, however, to regard it as the individual - a view which cannot be consistent in those cases where a simple or branched filamentous series exhibits differences between free apex and fixed base and so forth. It may be doubted whether the discussion is profitable, though it appears necessary in some cases - e.g. concerning pleomorphy - to adopt some definition of individual.

Myxobacteriaceae

To the two divisions of bacteria, Haplobacterinae and Trichobacterinae, must now be added a third division, Myxobacterinae. One of the first members of this group, Chondromyces crocatus, was described as long ago as 1857 by Berkeley, but its nature was not understood and it was ascribed to the Hyphomycetes. In 1892, however, Thaxter rediscovered it and showed its bacterial nature, founding for it and some allied forms the group Myxobacteriaceae. Another form, which he described as Myxobacter, was shown later to be the same as Polyangium vitellinum described by Link in 1795, the exact nature of which had hitherto been in doubt. Thaxter's observations and conclusions were called in question by some botanists, but his later observations and those of Baur have established firmly the position of the group. The peculiarity of the group lies in the fact that the bacteria form plasmodiumlike aggregations ' and build themselves up into sporogenous structures of definite form superficially similar to the cysts of the Mycetozoa (fig. 12). Most of the forms in question are found growing on the dung of herbivorous animals, but the bacteria occur not only in the alimentary canal of the animal but also free in the air. The Myxobacteria are most easily obtained by keeping at a temperature of 30-35° C. in the dark dung which has lain exposed to the air for at least eight days. The high temperature is favourable to the growth of the bacteria but FIG. 12.

A. Myxococcus digelatus,bright red fructification occurring on dung (X 120).

B. Polyangium primigenum, red fructification on dog's dung (X40)C. Chondromyces apiculatus, orange fructification on antelope's dung.

D. Young fructification (X45)E. Single cyst germinating (X200).

(A, B. after Quehl; C-E,after Thaxter.) From Strasburger's Lehrbuch der Bolanik, by permission of Gustav Fischer.

inimical to that of the fungi which are so common on this substratum.

The discoveries that some species of nitrifying bacteria and perhaps pigmented forms are capable of carbon-assimilation, that others can fix free nitrogen and that a number of decompositions hitherto unsuspected are accom fished by Schizomycetes have ut thequestions of P Y Y, P d nutrition and fermentation in quite new lights. Apart from numerous fermentation processes such as rotting, the soaking of skins for tanning, the preparation of indigo and of tobacco, hay, ensilage, &c., in all of which bacterial fermentations are concerned, attention may be especially directed to the following evidence of the supreme importance of Schizomycetes in agriculture and daily life. Indeed, nothing marks the attitude of modern bacteriology more clearly than the increasing attention which is being paid to useful fermentations. The vast majority of these organisms are not pathogenic, most are harmless and 4.,52 5.20 5.20 FIG. 13, - A series of phases of germination of the spore of B. ramosus sown at 8.30 (to the extreme left), showing how the growth can be measured. If we place the base of the filament in each case on a base line in the order of the successive times of observation recorded, and at distances apart proportional to the intervals of time (8.30, 10.0, 10.30, 11.40, and so on) and erect the straightened-out filaments, the proportional length of each of which is here given for each period, a line joining the tips of the filaments gives the curve of growth. (H. M. W.) many are indispensable aids in natural operations important to man.

Fischer has proposed that the old division into saprophytes and parasites should be replaced by one which takes into account other peculiarities in the mode of nutrition of bacteria. The nitrifying, nitrogen-fixing, sulphurand iron-bacteria he regards as monotrophic, i.e. as able to carry on one particular series of fermentations or decompositions only, and since they require no organic food materials, or at least are able to work up nitrogen or carbon from inorganic sources, he regards them as primitive forms in this respect and terms them Prototrophic. They may be looked upon as the nearest existing representatives of the primary forms of life which first obtained the power of working up non-living into living materials, and as playing a correspondingly important role in the evolution of life on our globe. The vast majority of bacteria, on the other hand, which are ordinarily termed saprophytes, are saprogenic, i.e. bring organic material to the putrefactive state - or saprophilous, i.e. live best in such putrefying materials - or become zymogenic, i.e. their metabolic products may induce blood-poisoning or other toxic effects (facultative parasites) though they are not true parasites. These forms are termed by Fischer Metatrophic, because they require various kinds of organic materials obtained from the dead remains of other organisms or from the surfaces of their bodies, and can utilize and decompose them in various ways (Polytrophic) or, if monotrophic, are at least unable to work them up. The true parasites - obligate parasites of de Bary - are placed by Fischer in a third biological group, Paratrophic bacteria, to mark the importance of their mode of life in the interior of living organisms where they live and multiply in the blood, juices or tissues.

When we reflect that some hundreds of thousands of tons of urea are daily deposited, which ordinary plants are unable to assimilate until considerable changes have been undergone, the question is of importance, What happens in the meantime? In effect the urea first becomes carbonate of ammonia by a simple hydrolysis brought about by bacteria, more and more definitely known since Pasteur, van Tieghem and Cohn first described them. Lea and Miguel further proved that the hydrolysis is due to an enzyme - urase - separable with difficulty from the bacteria concerned. Many forms in rivers, soil, manure heaps, &c., are capable of bringing about this change to ammonium carbonate, and much of the loss of volatile ammonia on farms is preventible if the facts are apprehended. The excreta of urea alone thus afford to the soil enormous stores of nitrogen combined in a form which can be rendered available by bacteria, and there are in addition the supplies brought down in rain from the atmosphere, and those due to other living debris. The researches of later years have demonstrated that a still more inexhaustible supply of nitrogen is made available by the nitrogen-fixing bacteria of the soil. There are in all cultivated soils forms of bacteria which are capable of forcing the inert free nitrogen to combine with other elements into compounds assimilable by plants. This was long asserted as probable before Winogradsky showed that the conclusions of M. P. E. Berthelot, A. Laurent and others were right, and that Clostridium pasteurianum, for instance, if protected from access of free oxygen by an envelope of aerobic bacteria or fungi, and provided with the carbohydrates and minerals necessary for its growth, fixes nitrogen in proportion to the amount of sugar consumed. This interesting case of symbiosis is equalled by yet another case. The work of numerous observers has shown that the free nitrogen of the atmosphere is brought into combination in the soil in the nodules filled with bacteria on the roots of Leguminosae, and since these nodules are the morphological expression of a symbiosis between the higher plant and the bacteria, there is evidently here a case similar to the last.

As regards the ammonium carbonate accumulating in the soil from the conversion of urea and other sources, we know from Winogradsky's researches that it undergoes oxidation in two stages owing to the activity of the so-called " nitrifying " bacteria (an unfortunate term inasmuch as " nitrification " refers merely to a particular phase of the cycle of changes undergone by nitrogen). It had long been known that under certain conditions large quantities of nitrate (saltpetre) are formed on exposed heaps of manure, &c., and it was supposed that direct oxidation of the ammonia, facilitated by the presence of porous bodies, brought this to pass. But research showed that this process of nitrification is dependent on temperature, aeration and moisture, as is life, and that while nitre-beds can infect one another, the process is stopped by sterilization. R. Warington, J. T. Schloessing, C. A. Mintz and others had proved that nitrification was promoted by some organism, when Winogradsky hit on the happy idea of isolating the organism by using gelatinous silica, and so avoiding the difficulties which Warington had shown to exist with the organism in presence of organic nitrogen, owing to its refusal to nitrify on gelatine or other nitrogenous media. Winogradsky's investigations resulted in the discovery that two kinds of bacteria are concerned in. nitrification; one of these, which he terms the Nitroso-bacteria., is only capable of bringing about the oxidation of the ammonia to nitrous acid, and the astonishing result was obtained that 12.42.1140, 10.01 10;3U 2.13, 2.35 2.58 4.52 3.43 :` 4.3 0 4.12 this can be done, in the dark, by bacteria to which only pure mineral salts - e.g. carbonates, sulphates and chlorides of ammonium, sodium and magnesium - were added. In other words these bacteria can build up organic matter from purely mineral sources by assimilating carbon from carbon dioxide in the dark and by obtaining their nitrogen from ammonia. The energy liberated during the oxidation of the nitrogen is regarded as splitting the carbon dioxide molecule, - in green plants it is the energy of the solar rays which does this. Since the supply of free oxygen is dependent on the activity of green plants the process is indirectly dependent on energy derived from the sun, but it is none the less an astounding one and outside the limits of our previous generalizations. It has been suggested that urea is formed by polymerization of ammonium carbonate, and formic aldehyde is synthesized from CO 2 and 011 2. The Nitro-bacteria are smaller, finer and quite different from the nitroso-bacteria, and are incapable of attacking and utilizing ammonium carbonate. When the latter have oxidized ammonia to nitrite, however, the former step in and oxidize it still further to nitric acid. It is probable that important consequences of these actions result from the presence of nitrifying bacteria in rotten stone, FIG. 14. - Stages in the formation of a colony of a variety of Bacillus (Proteus) vulgaris (Hauser), observed in a hanging drop. At I I A.M. a rodlet appeared (A); at 4 P.M. it had grown and divided and broken up into eight rodlets (B); C shows further development at 8 P.M., D at 9.30 P.M. - all under a high power. At E, F, and G further stages are drawn, as seen under much lower power. (H. M. W.) decaying bricks, &c., where all the conditions are realized for preparing primitive soil, the breaking up of the mineral constituents being a secondary matter. That " soil " is thus prepared on barren rocks and mountain peaks may be concluded with some certainty.

In addition to the bacterial actions which result in the oxidization of ammonia to nitrous acid, and of the latter to nitric acid, the reversal of such processes is also brought about by numerous bacteria in the soil, rivers, &c. Warington showed some time ago that many species are able to reduce nitrates to nitrites, and such reduction is now known to occur very widely in nature. The researches of Gayon and Dupetit, Giltay and Aberson and others have shown, moreover, that bacteria exist which carry such reduction still further, so that ammonia or even free nitrogen may escape. The importance of these results is evident in explaining an old puzzle in agriculture, viz. that it is a wasteful process to put nitrates and manure together on the land. Fresh manure abounds in de-nitrifying bacteria, and these organisms not only reduce the nitrates to nitrites, even setting free nitrogen and ammonia, but their effect extends to the undoing of the work of what nitrifying bacteria may be present also, with great loss. The combined nitrogen of dead organisms, broken down to ammonia by putrefactive bacteria, the ammonia of urea and the results of the fixation of free nitrogen, together with traces of nitrogen salts due to meteoric activity, are thus seen to undergo various vicissitudes in the soil, rivers and surface of the globe generally. The ammonia may be oxidized to nitrites and nitrates, and then pass into the higher plants and be worked up into proteids, and so be handed on to animals, eventually to be broken down by bacterial action again to ammonia; or the nitrates may be degraded to nitrites and even to free nitrogen or ammonia, which escapes.

That the Leguminosae (a group of plants including peas, beans, vetches, lupins, &c.) play a special part in agriculture was known even to the ancients and was mentioned by Pliny (Historia Naturalis, viii.). These plants will not only grow on poor sandy soil without any addition of nitrogenous manure, but they actually enrich the soil on which they are grown. Hence leguminous plants are essential in all rotation of crops. By analysis it was shown by Schulz-Lupitz in 1881 that the way in which these plants enrich the soil is by increasing the nitrogen-content. Soil which had been cultivated for many years as pasture was sown with lupins for fifteen years in succession; an analysis then showed that the soil contained more than three times as much nitrogen as at the beginning of the experiment. The only possible source for this increase was the atmospheric nitrogen. It had been, however, an axiom with botanists that the green plants were unable to use the nitrogen of the air. The apparent contradiction was explained by the experiments of H. Hellriegel and Wilfarth in 1888. They showed that, when grown on sterilized sand with the addition of mineral salts, the Leguminosae were no more able to use the atmospheric nitrogen than other plants such as oats and barley. Both kinds of plants required the addition of nitrates to the soil. But if a little water in which arable soil had been shaken up was added to the sand, then the leguminous plants flourished in the absence of nitrates and showed an increase in nitrogenous material. They had clearly made use of the nitrogen of the air. When these plants were examined they had small swellings or nodules on their roots, while those grown in sterile sand without soil-extract had no nodules. Now these peculiar nodules are a normal characteristic of the roots of leguminous plants grown in ordinary soil. The experiments above mentioned made clear for the first time the nature and activity of these nodules. They are clearly the result of infection (if the soil extract was boiled before addition to the sand no nodules were produced), and their presence enabled the plant to absorb the free nitrogen of the air. The work of recent investigators has made clear the whole process. In ordinary arable soil there exist motile rod-like bacteria, Bacterium radicicola. These enter the root-hairs of leguminous plants, and passing down the hair in the form of a long, slimy (zoogloea) thread, penetrate the tissues of the root. As a result the tissues become hypertrophied, producing the well-known nodule. In the cells of the nodule the bacteria multiply and develop, drawing material from their host. Many of the bacteria exhibit curious involution forms (" bacteroids "), which are finally broken down and their products absorbed by the plant. The nitrogen of the air is absorbed by the nodules, being built up into the bacterial cell and later handed on to the host plant. It appears from the observations of Maze that the bacterium can even absorb free nitrogen when grown in cultures FIG. 15. - Invasion of leguminous roots by bacteria.

cell from the epidermis of root of Pea with " infection thread " (zoogloea) pushing its way through the cellwalls. (After Prazmowski.) free end of a root-hair of Pea; at the right are particles of earth and on the left a mass of bacteria. Inside the hair the bacteria are pushing their way up in a thin stream.

(From Fischer's Vorlesungen uber Bakk rien.) outside the plant. We have here a very interesting case of symbiosis as mentioned above. The green plant, however, always keeps the upper hand, restricting the development of the bacteria to the nodules and later absorbing them for its own use. It should be mentioned that different genera require different races of the bacterium for the production of nodules.

The important part that these bacteria play in agriculture led to the introduction in Germany of a commercial product (the socalled " nitragin ") consisting of a pure culture of the bacteria, which is to be sprayed over the soil or applied to the seeds before sowing. This material was found at first to have a very uncertain effect, but later experiments in America, and the use of a modified preparation in England, under the direction of Professor Bottomley, have had successful results; it is possible that in the future a preparation of this sort will be widely used.

ization of these bacteria to the leguminous plants has always been a very striking fact, for similar bacterial nodules are known only in two or three cases out _ side this particular /01 group. However, Pro fessor Bottomley an nounced at the meeting of the British Association for the Advancement of Science in 1907 that he had succeeded in breaking down this specialization and by a suitable treatment had caused bacteria from leguminous nodules to infect other plants such as cereals, tomato, rose, with a marked effect on their growth. If these results are confirmed and the treatment can be worked commercially, the importance to agriculture of the discovery cannot be overestimated; each plant will provide, like the bean and vetch, its own nitrogenous manure, and larger crops will be produced at a decreased cost.

Another important advance is in our knowledge of the part played by bacteria in the circulation of carbon in nature. The enormous masses of cellulose deposited annually on the earth's surface are, as we know,! principally the result of chlorophyll action on the carbon dioxide of the atmosphere decomposed by energy derived from the sun; and although we know little as yet concerning the magnitude of other processes of carbon-assimilation - e.g. by nitrifying bacteria - it is probably comparatively small. Such cellulose is gradually reconverted into water and carbon dioxide, but for some time nothing positive was known as to the agents which thus break up the paper, rags, straw, leaves and wood, &c., accumulating in cesspools, forests, marshes and elsewhere in such abundance. The work of van Tieghem, van Senus, Fribes, Omeliansky and others has now shown that while certain anaerobic bacteria decompose the substance of the middle lamella - chiefly pectin compounds - and thus bring about the isolation of the cellulose fibres when, for instance, flax is steeped or " retted," they are unable to attack the cellulose itself. There exist in the mud of marshes, rivers and cloacae, &c., however, other anaerobic bacteria which decompose cellulose, probably hydrolysing it first and then splitting the products into carbon dioxide and marsh gas. When calcium sulphate is present, the nascent methane induces the formation of calcium carbonate, sulphuretted hydrogen and water. We have thus an explanation of the occurrence of marsh gas and sulphuretted hydrogen in bogs, and it is highly probable that the existence of these gases in the intestines of herbivorous animals is due to similar putrefactive changes in the undigested cellulose remains.

Cohn long ago showed that certain glistening particles observed in the cells of Beggiatoa consist of sulphur, and Winogradsky and Beyerinck have shown that a whole series of sulphur bacteria of the genera Thiothrix, Chromatium, Spirillum, Monas, &c., exist, and play important parts in the circulation of this element in nature, e.g. in marshes, estuaries, sulphur springs, &c. When cellulose bacteria set free FIG. 17. - A plate-culture of a bacillus which had been exposed for a period of four hours behind a zinc stencil-plate, in which the letters C and B were cut. The light had to traverse a screen of water before passing through the C, and one of aesculin (which filters out the blue and violet rays) before passing the B. The plate was then incubated, and, as the figure shows, the bacteria on the C-shaped area were all killed, whereas they developed elsewhere on the plate (traces of the B are just visible to the right) and covered it with an opaque growth. (H. M. W.) marsh gas, the nascent gas reduces sulphates - e.g. gypsum - with liberation of SH 2, and it is found that the sulphur bacteria thrive under such conditions by oxidizing the SH 2 and storing the sulphur in their own protoplasm. If the SH 2 runs short they oxidize the sulphur again to sulphuric acid, which combines with any calcium carbonate present and forms sulphate again. Similarly nascent methane may reduce iron salts, and the black mud in which these bacteria often occur owes its colour to the FeS formed. Beyerinck and Jegunow have shown that some partially anaerobic sulphur bacteria can only exist in strata at a certain depth below the level of quiet waters where SH 2 is being set free below by the bacterial decompositions of vegetable mud and rises to meet the atmospheric oxygen coming down from above, and that this zone of physiological activity rises and falls with the variations of partial pressure of the gases due to the rate of evolution of the SH 2. In the deeper parts of this zone the bacteria absorb the SH 2, and, as they rise, oxidize it and store up the sulphur; then ascending into planes more highly oxygenated, oxidize the sulphur to SO 3. These bacteria therefore employ SH 2 as their respiratory substance, much as higher plants employ carbohydrates - instead of liberating energy as heat by the respiratory combustion of sugars, they do it by oxidizing hydrogen sulphide. Beyerinck has shown that Spirillum desulphuricans, a definite anaerobic form, attacks and reduces sulphates, thus undoing the work of the sulphur bacteria as certain de-nitrifying bacteria reverse the operations of nitro-bacteria. Here again, therefore, we have sulphur, taken FIG. 16.

a, root nodule of the lupin, nat. size. (From Woromv.) b, longitudinal section through root and nodule.

g, fibro-vascular bundle.

w, bacterial tissue. (After Woromv.) c, cell from bacterial tissues showing nucleus and protoplasm filled with bacteria.

d, bacteria from nodule of lupin, normal undegenerate form.

e and f, bacteroids from Vicia villosa and Lupinus albus. (After Morck. (c X 600;) al X 1500, above.) (From Fischer's Vorlesungen uber Bakterien.) into the higher plants as sulphates, built up into proteids, decomposed by putrefactive bacteria and yielding SH 2 which the sulphur bacteria oxidize; the resulting sulphur is then again oxidized to SO 3 and again combined with calcium to gypsum, the cycle being thus complete.

Chalybeate waters, pools in marshes near irons one, &c., abound in bacteria,, some of which belong to the remarkable genera Crenothrix, Cladothrix and Leptothrix, and contain ferric oxide, i.e. rust, in their cell-walls. This iron deposit is not merely mechanical but is due to the physiological activity of the organism which, according to Winogradsky, liberates energy by oxidizing ferrous and ferric oxide in its protoplasm - a view not accepted by H. Molisch. The iron must be in certain soluble conditions, however, and the soluble bicarbonate of the protoxide of chalybeate springs seems most favourable; the hydrocarbonate absorbed by the cells is oxidized, probably thus 2FeCO 3 1-30H 2 +O = Fee (OH)6+2C02.

The ferric hydroxide accumulates in the sheath, and gradually passes into the more insoluble ferric oxide. These actions are of extreme importance in nature, as their continuation results in the enormous deposits of bog-iron ore, ochre, and - since Molisch has shown that the iron can be replaced by manganese in some bacteria - of manganese ores.

Considerable advances in our knowledge of the various chromogenic bacteria have been made by the studies of Beyerinck, Lankester, Engelmann, Ewart and others, and have assumed exceptional importance owing to the discovery that Bacteriopurpurin - the red colouring matter contained in certain sulphur bacteria - absorbs certain rays of solar energy, and enables the organism to utilize the energy for its own life-purposes. Engelmann showed, for instance, that these red-purple bacteria collect in the ultra-red, and to a less extent in the orange and green, in bands which agree with the absorption spectrum of the extracted colouring matter. Not only so, but the evident parallelism between this absorption of light and that by the chlorophyll of green plants, is completed by the demonstration that oxygen is set free by these bacteria - i.e. by means of radiant energy trapped by their colour-screens the living cells are in both cases enabled to do work, such as the reduction of highly oxidized compounds.

The most recent observations of Molisch seem to show that bacteria possessing bac eriopurpurin exhibit a new type of assimilation - the assimilation of organic material under the influence of light. In the case of these red-purple bacteria the colouring matter is contained in the protoplasm of the cell, but in most chromogenic bacteria it occurs as excreted pigment on and between the cells, or is formed by their action in the medium. Ewart has confirmed the principal conclusions concerning these purple, and also the so-called chlorophyll bacteria (B. viride, B. chlorinum, &c.), the results going to show that these are, as many authorities have held, merely minute algae. The pigment itself may be soluble in water, as is the case with the blue-green fluorescent body formed by B. pyocyaneus, B. fluorescens and a whole group of fluorescent bacteria. Neelson found that the pigment of B. cyanogenus gives a band in the yellow and strong lines at E and F in the solar spectrum - an absorption spectrum almost identical with that of triphenyl-rosaniline. In the case of the scarlet and crimson red pigments of B. prodigiosus, B. ruber, &c., the violet of B. violacens, B. janthinus, &c., the redpurple of the sulphur bacteria, and indeed most bacterial pigments, solution in water does not occur, though alcohol extracts the colour readily. Finally, there are a few forms which yield their colour to neither alcohol nor water, e.g. the yellow Micrococcus cereus-flavus and the B. berolinensis. Much work is still necessary before we can estimate the importance of these pigments. Their spectra are only imperfectly known in a few cases, and the bearing of the absorption on the life-history is still a mystery. In many cases the colour-production is dependent on certain definite conditions - temperature, presence of oxygen, nature of the food-medium, &c. Ewart's important discovery that some of these lipochrome pigments occlude oxygen, while others do not, may have bearings on the facultative anaerobism of these organisms.

A branch of bacteriology which offers numerous problems of importance is that which deals with the organisms so common in milk, butter and cheese. Milk is a medium not only admirably suited to the growth of bacteria, but ry Y Y g as a matter of fact, always contaminated with these organisms in the ordinary course of supply. F. Lafar has stated that 20% of the cows in Germany suffer from tuberculosis, which also affected 17.7% of the cattle slaughtered in Copenhagen between 1891 and 1893, and that one in every thirteen samples of milk examined in Paris, and one in every nineteen in Washington, contained tubercle bacilli. Hence the desirability of sterilizing milk used for domestic purposes becomes imperative.

FIG. 18. - A similar preparation to fig. 17, except that two slit-like openings of equal length allowed the light to pass, and that the light was that of the electric arc passed through a quartz prism and casting a powerful spectrum on the plate. The upper slit was covered with glass, the lower with quartz. The bacteria were killed over the clear areas shown. The left-hand boundary of the clear area corresponds to the line F (green end of the blue), and the beginning of the ultra-violet was at the extreme right of the upper (short) area. The lower area of bactericidal action extends much farther to the right, because the quartz allows more ultra-violet rays to pass than does glass. The red-yellow-green to the left of F were without effect. (H. M. W.) No milk is free from bacteria, because the external orifices of the milk-ducts always contain them, but the forms present in the normal fluid are principally those which induce such changes as the souring or " turning " so frequently observed in standing milk (these were examined by Lord Lister as long ago as 1873-1877, though several other species are now known), and those which bring about the various changes and fermentations in butter and cheese made from it. The presence of foreign germs, which may gain the upper hand and totally destroy the flavours of butter and cheese, has led to the search for those particular forms to which the approved properties are due. A definite bacillus to which the peculiarly fine flavour of certain butters is due, is said to be largely employed in pure cultures in American dairies, and in Denmark certain butters are said to keep fresh much longer owing to the use of pure cultures and the treatment employed to suppress the forms which cause rancidity. Quite distinct is the search for the germs which cause undesirable changes, or " diseases "; and great strides have been made in discovering the bacteria concerned in rendering milk " ropy," butter " oily " and " rancid," &c. Cheese in its numerous forms contains myriads of bacteria, and some of these are now known to be concerned in the various processes of ripening and other changes affecting the product, and although little is known as to the exact part played by any species, practical applications of the discoveries of the decade 1890-1900 have been made, e.g. Edam cheese. The Japanese have cheeses resulting from the bacterial fermentation of boiled Soja beans.

cultivation of which have been successfully carried out by Cohn, Beyerinck, Fischer and others. The cause of the phosphorescence is still a mystery. The suggestion that it is due to the oxidation of a body excreted by the bacteria seems answered by the failure to filter off or extract any such body. Beyerinck's view that it occurs at the moment peptones are worked up into the protoplasm cannot be regarded as proved, and the same must be said of the suggestion that the phosphorescence is due to the oxidation of phosphoretted hydrogen. The conditions of phosphorescence are, the presence of free oxygen, and, generally, a relatively low temperature, together with a medium containing sodium chloride, and peptones, but little or no carbohydrates. Considerable differences occur in these latter respects, however, and interesting results were obtained by Beyerinck with mixtures of species possessing different powers of enzyme action as regards carbohydrates. Thus, a form termed Photobacterium phosphorescens by Beyerinck will absorb maltose, and will become luminous if that sugar is present, whereas P. Pflugeri is indifferent to maltose. If then we prepare densely inseminated plates of these two bacteria in gelatine food-medium to which starch is added as the only carbohydrate, the bacteria grow but do not phosphoresce. If we now streak these plates with an organism, e.g. a yeast, which saccharifies starch, it is possible to tell whether maltose or levulose and fructose are formed; if the former, only those plates containing P. phosphorescens will become luminous; if the latter, only those containing P. Pflugeri. The more recent researches of Molisch have shown that the luminosity of ordinary butcher's meat under appropriate conditions is quite a common occurrence. Thus of samples of meat bought in Prague and kept in a cool room for about two days, luminosity was present in 52% of the samples in the case of beef, 50% for veal, and 39% for liver. If the meat was treated previously with a 3% salt solution, 89% of the samples of beef and 65% of the samples of horseflesh were found to exhibit this phenomenon. The cause of this luminosity is Micrococcus phosphorens, an immotile round, or almost round organism. This organism is quite distinct from that causing the luminosity of marine fish.

It has long been known that the production of vinegar depends on the oxidization of the alcohol in wine or beer to acetic acid, the chemical process being probably carried out in two stages, viz. the oxidation of the alcohol leading to the formation of aldehyde and water, and the further oxidation of the aldehyde to acetic acid. The process may even go farther, and the acetic acid be oxidized to CO 2 and 01{2; the art of the vinegar-maker is directed to preventing the accomplishment of the last stage. These oxidations are brought about by the vital activity of several bacteria, of which four-- Bacterium aceti, B. pasteurianum, B. kiitzingianum, and B. xylinum - have been thoroughly studied by Hansen and A. Brown. It is these bacteria which form the zoogloea of the " mother of vinegar," though this film may contain other organisms as well. The idea that this film of bacteria oxidizes the alcohol beneath by merely condensing atmospheric oxygen in its interstices, after the manner of spongy platinum, has long been given up; but the explanation of the action as an incomplete combustion, depending on the peculiar respiration of these organisms - much as in the case of nitrifying and sulphur bacteria - is not clear, though the discovery that the acetic bacteria will not only oxidize alcohol to acetic acid, but further oxidize the latter to CO 2 and 01-1 2 supports the view that the alcohol is absorbed by the organism and employed as its respirable substance. Promise of more light on these oxidation fermentations is afforded by the recent discovery that not only bacteria and fungi, but even the living cells of higher plants, contain peculiar enzymes which possess the remarkable property of " carrying " oxygen - much as it is carried in the sulphuric acid chamber - and which have therefore been termed oxydases. It is apparently the presence of these oxydases which causes certain wines to change colour and alter in taste when poured from bottle to glass, and so exposed to air.

Much as the decade from 1880 to 1890 abounded with investigations on the reactions of bacteria to heat, so the following decade was remarkable for discoveries regarding the effects of other forms of radiant energy. The observations Bacteria g 5'of Downes and Blunt in 1877 left it uncertain whether the bactericidal effects in broth cultures exposed to solar rays were due to thermal action or not. Further investigations, in FIG. 19. - Ginger-beer plant, showing yeast (Saccharomyces pyrifor7nis) entangled in the meshes of the bacterium (B. vermiforme). (H. M. W.) which Arloing, Buchner, Chmelewski, and others took part, have led to the proof that rays of light alone are quite capable of killing these organisms. The principal questions were satisfactorily settled by Marshall Ward's experiments in 1892-1893, when he showed that even the spores of B. anthraces, which withstand temperatures of loo C. and upwards, can be killed by exposure to rays of reflected light at temperatures far below anything injurious, or even favourable to growth. He also showed that the bactericidal action takes place in the absence of food materials, thus proving that it is not merely a poisoning effect of the altered medium. The principal experiments also indicate that it is the rays of highest refrangibility - the blue-violet and ultra-violet rays of the spectrum - which bring about the destruction of the organisms (figs. 17,18). The practical effect of the bactericidal action of solar light is the destruction of enormous quantities of germs in rivers, the atmosphere and other exposed situations, and experiments have shown that it is especially the pathogenic bacteria - anthrax, typhoid, &c. - which thus succumb to lightaction; the discovery that the electric arc is very rich in bactericidal rays led to the hope that it could be used for disinfecting purposes in hospitals, but mechanical difficulties intervene. The recent application of the action of bactericidal rays to the cure of lupus is, however, an extension of the same discovery. Even when the light is not sufficiently intense, or the exposure is too short to kill the spores, the experiments show that attenuation of virulence, That bacterial fermentations are accompanied by the evolution of heat is an old experience; but the discovery that the " spontaneous " combustion of sterilized cotton-waste does not occur simply if moist and freely exposed to oxygen, philous bacteria. but results when the washings of fresh waste are added, has led to clearer proof that the heating of hay-stacks, hops, tobacco and other vegetable products is due to the vital activity of bacteria and fungi, and is physiologically a consequence of respiratory processes like those in malting. It seems fairly established that when the preliminary heating process of fermentation is drawing to a close, the cotton, hay, &c., having been converted into a highly porous friable and combustible mass, may then ignite in certain circumstances by the occlusion of oxygen, just as ignition is induced by finely divided metals.

A remarkable point in this connexion has always been the necessary conclusion that the living bacteria concerned must be exposed to temperatures of at least 70° C. in the hot heaps. Apart from the resolution of doubts as to the power of spores to withstand such temperatures for long periods, the discoveries of Miguel, Globig and others have shown that there are numerous bacteria which will grow and divide at such temperatures, e.g.

B. thermophilus, from sewage, which is quite active at 70 0 C., and B. Ludwigi and B. ilidzensis, &c., from hot springs, &c.

The bodies of sea fish, e.g. mackerel and other animals, have long been known to exhibit phosphorescence. This phenomenon is due to the activity of a whole series of marine bacteria of various genera, the examination and organisms depend on the discovery that their patho genicity or virulence can be modified - diminished or increased - by definite treatment, and, in the natural course of epidemics, by alterations in the environment. Similarly we are unable to divide Schizomycetes sharply into parasites and saprophytes, since it is well proved that a number of species - facultative parasites - can become one or the other according to circumstances. These facts, and the further knowledge that many bacteria never observed as parasites, or as pathogenic forms, produce toxins or poisons as the result of their decompositions and fermentations of organic substances, have led to important results in the applications of bacteriology to medicine. Bacterial diseases in the higher plants have been described, but the subject requires careful treatment, since several points suggest doubts as to the organism described being the of the disease referred to their agency. Until g ?' recently it was urged that the acid contents of plants explained their immunity from bacterial diseases, but it is now known that many bacteria can flourish in acid media. Another objection was that even if bacteria obtained access through the stomata, they could not penetrate the cell-walls bounding the intercellular spaces, but certain anaerobic forms are known to ferment cellulose, and others possess the power of penetrating the cell-walls of living cells, as the bacteria of Leguminosae first described by Marshall Ward in 1887, and confirmed by Miss Dawson in 1898. On the other hand a long list of plant-diseases has been of late years attributed to bacterial action. Some, e.g. the Sereh disease of the sugar-cane, the slime fluxes of oaks and other trees, are not only very doubtful cases, in which other organisms such as yeasts and fungi play their parts, but it may be regarded as extremely improbable that the bacteria are the primary agents at all; they are doubtless saprophytic forms which have gained access to rotting tissues injured by other agents. Saprophytic bacteria can readily make their way down the dead hypha of an invading fungus, or into the punctures made by insects, and Aphides have been credited with the bacterial infection of carnations, though more recent researches by Woods go to show the correctness of his conclusion that Aphides alone are responsible for the carnation disease. On the other hand, recent investigation has brought to light cases in which bacteria are certainly the primary agents in diseases of plants. The principal features are the stoppage of the vessels and consequent wilting of the shoots; as a rule the cut vessels on transverse sections of the shoots appear brown and choked with a dark yellowish slime in which bacteria may be detected, e.g. cabbages, cucumbers, potatoes, &c. In the carnation disease and in certain diseases of tobacco and other plants the seat of bacterial action appears to be the parenchyma, and it may be that Aphides or other piercing insects infect the plants, much as insects convey pollen from plant to plant, or (though in a different way) as mosquitoes infect man with malaria. If the recent work on the cabbage disease may be accepted, the bacteria make their entry at the water pores at the margins of the leaf, and thence via the glandular cells to the tracheids. Little is known of the mode of action of bacteria on these plants, but it may be assumed with great confidence that they excrete enzymes and poisons (toxins), which diffuse into the cells and kill them, and that the effects are in principle the same as those of parasitic fungi. Support is found for this opinion in Beyerinek's discovery that the juices of tobacco plants affected with the disease known as " leaf mosaic," will induce this disease after filtration through porcelain.

In addition to such cases as the kephir and ginger-beer plants (figs. 19, 20), where anaerobic bacteria are associated with yeasts, several interesting examples of symbiosis among bacteria are now known. Bacillus chauvaei ferments cane-sugar solutions in such a way that normal butyric acid, inactive lactic acid, carbon dioxide, and FIG. 20. - The ginger-beer plant.

A. One of the brain-like gelatinous masses into which the mature condenses.

B. The bacterium with and without its gelatinous sheaths (cf. fig. 19).

C. Typical filaments and rodlets in the slimy sheaths.

D. Stages of growth of a sheathed filament - a at 9 A.M., b at 3 P.M., cat 9 P.M., d at I I A.M. next day, e at 3 P.M., fat 9 P.M., g at Io.30 A.M. next day, h at 24 hours later. (H. M. W.) hydrogen result; Micrococcus acidi-paralactici, on the other hand, ferments such solutions to optically active paralactic acid. Nencki showed, however, that if both these organisms occur together, the resulting products contain large quantities of normal butyl alcohol, a substance neither bacterium can produce alone. Other observers have brought forward other cases. Thus neither B. coli nor the B. denitrificans of Burri and Stutzer can reduce nitrates, but if acting together they so completely undo the structure of sodium nitrate that the nitrogen passes off in the free state. Van Senus showed that the concurrence of two bacteria is necessary before his B. amylobacter can ferment cellulose, and the case of mud bacteria which evolve sulphuretted hydrogen below which is utilized by sulphur bacteria above has already been quoted, as also that of Winogradsky's Clostridium III. 6 a may result, a point of extreme importance in connexion with the lighting and ventilation of dwellings, the purification of rivers and streams, and the general diminution of epidemics in nature.

As we have seen, thermophilous bacteria can grow at high temperatures, and it has long been known that some forms develop on ice. The somewhat different question of the resistance of ripe spores or cells to extremes of heat and cold has received attention. Ravenel, Macfadyen and Rowland have shown that several bacilli will bear exposure for seven days to the temperature of liquid air (- 192° C. to - 183° C.) and again grow when put into normal conditions. More recent experiments have shown that even ten hours' exposure to the temperature of liquid hydrogen - 252° C. (21° on the absolute scale) failed to kill them. It is probable that all these cases of resistance of seeds, spores, &c., are to be connected with the fact that completely dry albumin does not lose its coagulability on heating to I Io° C. for some hours, since it is well known that completely ripe spores and dry heat are the conditions of extreme experiments. No sharp line can be drawn between pathogenic and nonpathogenic Schizomycetes, and some of the most marked steps in the progress of our modern knowledge of these pasteurianum, which is anaerobic, and can fix nitrogen only if protected from oxygen by aerobic species. It is very probable that numerous symbiotic fermentations in the soil are due to this co-operation of oxygen-protecting species with anaerobic ones, e.g. Tetanus. Astonishment has been frequently expressed at the powerful activities of bacteria - their rapid growth and dissemination, of the extensive and profound decompositions and Activity bacteria. fermentations induced by them, the resistance of their spores to dessication, heat, &c. - but it is worth while to ask how far these properties are really remarkable when all the data for comparison with other organisms are considered. In the first place, the extremely small size and isolation of the vegetative cells place the protoplasmic contents in peculiarly favourable circumstances for action, and we may safely conclude that, weight for weight and molecule for molecule, the protoplasm of bacteria is brought into contact with the environment at far more points and over a far larger surface than is that of higher organisms, whether - as in plants - it is distributed in thin layers round the sap-vacuoles, or - as in animals - is bathed in fluids brought by special mechanisms to irrigate it. Not only so, the isolation of the cells facilitates the exchange of liquids and gases, the passage in of food materials and out of enzymes and products of metabolism, and thus each unit of protoplasm obtains opportunities of immediate action, the results of which are removed with equal. rapidity, not attainable FIG. 21. - A plate-culture colony of a in more complex multispecies of Bacillus - Proteus (Hauser) cellular organisms. To - on the fifth day. The flame-like P u t the matter in another processes and outliers are composed of way, if we could imagine writhing filaments, and the contours all the living cells of a are continually changing while the large oak or of a horse, colony moves as a whole. Slightly g magnified. (H. M. W.) having given up the specializations of func tion impressed on them during evolution and simply carrying out the fundamental functions of nutrition, growth, and multiplication which mark the generalized activities of the bacterial cell, and at the same time rendered as accessible to the environment by isolation and consequent extension of surface, we should doubtless find them exerting changes in the fermentable fluids necessary to their life similar to those exerted by an equal mass of bacteria, and that in proportion to their approximation in size to the latter. Ciliary movements, which undoubtedly contribute in bringing the surface into contact with larger supplies of oxygen and other fluids in unity of time, are not so rapid or so extensive when compared with other standards than the apparent dimensions of the microscopic field. The microscope magnifies the distance traversed as well as the organism, and although a bacterium which covers 9 - ro cm. or more in 15 minutes - say oi mm. or loo p, per second - appears to be darting across the field with great velocity, because its own small size - say 5 X i µ - comes into comparison, it should be borne in mind that if a mouse 2 in. long only, travelled twenty times its own length, i.e. 40 in., in a second, the distance traversed in 15 minutes at that rate, viz. 1000 yards, would not appear excessive. In a similar way we must be careful, in our wonder at the marvellous rapidity of cell-division and growth of bacteria, that we do not exaggerate the significance of the phenomenon. It takes any ordinary rodlet 30-40 minutes to double its length and divide into two equal daughter cells when growth is at its best; nearer the minimum it may require 3-4 hours or even much longer. It is by no means certain that even the higher rate is greater than that exhibited by a tropical bamboo which will grow over a foot a day, or even common grasses, or asparagus, during the active period of cell-division, though the phenomenon is here complicated by the phase of extension due to intercalation of water. The enormous extension of surface also facilitates the absorption of energy from the environment, and, to take one case only, it is impossible to doubt that some source of radiant energy must be at the disposal of those prototrophic forms which decompose carbonates and assimilate carbonic acid in the dark and oxidize nitrogen in dry rocky regions where no organic materials are at their disposal, even could they utilize them. is usually stated that the carbon dioxide molecule is here.

FIG. 22. - Portions of a colony such as that in fig. 2r, highly magnified, showing the kinds of changes brought about in a few minutes, from A to B, and B to C, by the growth and ciliary movements of the filaments. The arrows show the direction of motion. (H. M. W.) split by means of energy derived from the oxidation of nitrogen, but apart from the fact that none of these processes can proceed until the temperature rises to the minimum cardinal point, Engelmann's experiment shows that in the purple bacteria rays are used other than those employed by green plants, and especially ultra-red rays not seen in the spectrum, and we may probably conclude that " dark rays " - i.e. rays not appearing in the visible spectrum - are absorbed and employed by these and other colourless bacteria. The purple bacteria have thus two sources of energy, one by the oxidation of sulphur and another by the absorption of " dark rays." Stoney (Scient. Proc. R. Dub. Soc., 18 93, p. 1 54) has suggested yet another source of energy, in the bombardment of these minute masses by the molecules of the environment, the velocity of which is sufficient to drive them well into the organism, and carry energy in of which they can avail themselves.

Authorities

- General: Fischer, The Structure and Functions of Bacteria (Oxford, 1900, 2nd ed.), German (Jena, 1903); Migula, System der Bakterien (Jena, 1897); and in Engler and Prantl, Die natiirlichen Pflanzenfamilien, I. Th. I Abt. a; Lafar, Technical Mycology (vol. i. London, 1898); Mace, Traite pratique de bakteriologie (5th ed. 1904). Fossil bacteria: Renault, " Recherches sur les Bactbriacees fossiles," Ann. des Sc. Nat., 1896, p. 275. Bacteria in Water: Frankland and Marshall Ward. " Reports on the Bacteriology of Water," Proc. R. Soc. vol. li. p. 183, vol. liii. p. 245, vol. lvi. p. i; Marshall Ward, " On the Biology of B. ramosus," Proc. R. Soc., vol. lviii. p. 1; and papers on Bacteria of the river Thames in Ann. of Bot. vol. xii. pp. 59 and 287, and vol. xiii. p. 197. Cell-membrane, &c.: Biitschli, Weitere Ausfuhrungen uber den Bau der Cyanophyceen and Bakterien (Leipzig, 1896); Fischer, Unters. uber den Bau der Cyanophyceen and Bakterien (Jena, 1897); Rowland, " Observations upon the Structure of Bacteria," Trans. Jenner Institute, 2nd ser. 18 99, p. 1 43, with literature. Cilia: Fischer, " Unters. fiber Bakterien," Pringsh. Jahrb. vol. xxvii.; also the works of Migula and Fischer already cited. Nucleus: Wager in Ann. Bot. vol. ix. p. 659; also Migula and Fischer, l.c.; Vejdovsky, " fiber den Kern der Bakterien and seine Teilung," Cent. f. Bakt. Abt. II. Bd. xi. (1904) p. 481; ibid. " Cytologisches fiber die Bakterien der Prager Wasserleitung," Cent. f. Bakt. Abt. II. Bd. xv. (1905); Mend, " Nachtr ge zu den Strukturverhdltnissen von Bakterium gammari " in Archiv f. Protistenkunde, Bd. viii. (1907), p. 257. Spores, &c.: Marshall Ward, " On the Biology of B. ramosus," Proc. R. Soc., 1895, vol. lviii. p. r; Sturgis, " A Soil Bacillus of the type of de Bary's B. megatherium," Phil. Trans. vol. cxci. p. 147; Klein, L., Be y. d. deutschen hot. Gesellsch. (1889), Bd. vii.; and Cent. f. Bakt. and Par. (1889), Bd. vi. Classification: Marshall Ward, " On the Characters or Marks employed for classifying the Schizomycetes," Ann. of Bot., 1892, vol. vi.; Lehmann and Neumann, Atlas and Essentials of Bacteriology; also the works of Migula and Fischer already cited. Myxobacteriaceae: Berkeley, Introd. to Cryptogamic Botany (1857), p. 313; Thaxter, " A New Order of Schizomycetes," Bot. Gaz. vol. xvii. (1892), p. 389; and " Further Observations on the Myxobacteriaceae," ibid. vol. xxiii. (1897), p. 395, an d " Notes on the Myxobacteriaceae," ibid.vol. xxxvii. (1904), p. 405; Baur, " Myxobakterienstudien," Arch. f. Protistenkunde, Bd. v. (1904), p. 92; Smith, " Myxobacteria," Jour. of Botany, 1901, p. 69; Quehl, Cent. f. Bakt. xvi. (1896), p. 9. Growth: Marshall Ward, " On the Biology of B. ramosus," Proc. R. Soc. vol.

p. 1 (1895). Fermentation, &c.: Warington, The Chemical Action of some Micro-organisms (London, 1888); Winogradsky, " Recherches sur les organismes de la nitrification," Ann. de l'Inst. Past., 1890, pp. 213, 257, 760, 1891, pp. 92 and 577; " Sur l'assimilation de l'azote gazeux, &c.," Compt. Rend., 12 Feb. 1894; " Zur Microbiologie des Nitrifikationsprozesses," Cent. f. Bakt. Abt. II. Bd. ii. (1896), p. 415; " Ueber Schwefel-Bakterien," Bot. Zeitg., 1887, Nos. 31-37; Beitr. zur Morph. u. Phys. der Bakterien, H. i (1888); " Ueber Eisenbakterien," Bot. Zeitg., 1888, p. 261; and Omeliansky, " Ueber den Einfluss der organischen Substanzen auf die Arbeit der nitrifizierenden Organismen," Cent. f. Bakt. Abt. II. Bd. v. (1896); Schorler, " Beitr. zur Kenntniss der Eisenbakterien," Cent. f. Bakt. Abt. II. Bd. xii. (1904), p. 681; Marshall Ward, " On the Tubercular Swellings on the Roots of Vicia Faba," Phil. Trans., 1877, p. 539; Hellriegel and Wilfarth, " IJnters. fiber die Stickstoffnahrung der Gramineen u. Leguminosen," Beit. Zeit. d. Vereins filr die Ri benzuckerindustrie (Berlin, 1888); Nobbe and Hiltner, Landw. Versuchsstationen (1899), Bd. 51, p. 241, and Bd. 52, p. 455; Maze, Annales de l'Institut Pasteur, t. 11, p. 44, and t. 12, p. 1 (1897); Prazmowski, Land. Versuchsstationen, Bd. 37 (1890), p. 161, Bd. 38 (1891), p. 5; Frank, Landw. Jahrb. Bd. 17 (1888), P. 44 1; Omelianski, " Sur la fermentation de la cellulose," Compt. Rend., 4 Nov. 1895; van Senus, Beitr. zur Kenntn. der Cellulosegdhrung (Leiden, 1890); van Tieghem, " Sur la fermentation de la cellulose," Bull. de la soc. bot. de Fr. t.xxvi. (1879), p. 28; Beyerinck " Ueber Spirillum desulphuricans, &c.," Cent. f. Bakt. Abt. II. Bd. i. (1895), p. 1; Molisch, Die Pflanze in ihren Beziehungen zum Eisen (Jena, 1892). Pigment Bacteria: Ewart, " On the Eevolution of Oxygen from Coloured Bacteria," Linn. Journ., 1897, vol. xxxiii. p..123; Molisch, Die Purpurbakterien (Jena, 1907). Oxydases and Enzymes: Green, The Soluble Ferments and Fermentation (Cambridge, 1899). Action of Light, &c.: Marshall Ward, " The Action of Light on Bacteria," Phil. Trans., 18 93, p. 961, and literature. Resistance to Cold, &c.: Ravenel, Med. News, 1899, vol. lxxiv.; Macfadyen and Rowland, Proc. R. Soc. vol. lxvi. pp. 180, 339, and 488; Farmer, " Observations on the Effect of Desiccation of Albumin upon its Coagulability," ibid. p. 329. Pathogenic Bacteria: Baumgarten, Pathologische Mykologie (1890); Kolle and Wassermann, Handbuch der pathogenen Mikroorganismen (1902-1904); and numerous special works in medical literature. Immunity: Ehrlich, " On Immunity with Special Reference to Cell-life," Proc. R. Soc. vol. lxvi. p. 424; Calcar, " Die Fortschritte der Immunitatsund Spezifizetatslehre seit 1870," Progressus Rei Botanicae, Bd.

I. Heft 3(1907). Bacteriosis: Migula, l.c. p. 322, has collected the literature; see also Sorauer, Handbuch der Pflanzenkrankheiten, I. (1905), pp. 18-93, for later literature. Symbiosis: Marshall Ward, " Symbiosis," Ann. of Bot. vol. xiii. p. 549, and literature.

(H. M. W.; V. H. B.) Pathological Importance The action of bacteria as pathogenic agents is in great part merely an instance of their general action as producers of chemical change, yet bacteriology as a whole has become so extensive, and has so important a bearing on subjects widely different from one another, that division of it has become essential. The science will accordingly be treated in this section from the pathological standpoint only. It will be considered under the three following heads, viz. (1) the methods employed in the study; (2) the modes of action of bacteria and the effects produced by them; and (3) the facts and theories with regard to immunity against bacterial disease.

The demonstration by Pasteur that definite diseases could be produced by bacteria, proved a great stimulus to research in the etiology of infective conditions, and the result Historical was a rapid advance in human knowledge. An all important factor in this remarkable progress was the introduction by Koch of solid culture media, of the " platemethod," &c., an account of which he published in 1881. By means of these the modes of cultivation, and especially of separation, of bacteria were greatly simplified. Various modifications have since been made, but the routine methods in bacteriological procedure still employed are in great part those given by Koch. By 1876 the anthrax bacillus had been obtained in pure culture by Koch, and some other pathogenic bacteria had been observed in the tissues, but it was in the decade 1880-1890 that the most important discoveries were made in this field. Thus the organisms of suppuration, tubercle, glanders, diphtheria, typhoid fever, cholera, tetanus, and others were identified, and their relationship to the individual diseases established. In the last decade of the 19th century the chief discoveries were of the bacillus of influenza (1892), of the bacillus of plague (1894) and of the bacillus of dysentery (1898). Immunity against diseases caused by bacteria has been the subject of systematic research from 1880 onwards. In producing active immunity by the attenuated virus, Duguid and J. S. Burdon-Sanderson and W. S. Greenfield in Great Britain, and Pasteur, Toussaint and Chauveau in France, were pioneers. The work of Metchnikoff, dating from about 1884, has proved of high importance, his theory of phagocytosis (vide infra) having given a great stimulus to research, and having also contributed to important advances. The modes by which bacteria produce their effects also became a subject of study, and attention was naturally turned to their toxic products. The earlier work, notably that of L. Brieger, chiefly concerned ptomaines (vide infra), but no great advance resulted. A new field of inquiry was, however, opened up when, by filtration a bacterium-free toxic fluid was obtained which produced the important symptoms of the disease-in the case of diphtheria by P. P. E. Roux and A. Yersin (1888), and in the case of tetanus a little later by various observers. Research was thus directed towards ascertaining the nature of the toxic bodies in such a fluid, and Brieger and Fraenkel (1890) found that they were proteids, to which they gave the name " toxalbumins." Though subsequent researches have on the whole confirmed these results, it is still a matter of dispute whether these proteids are the true toxins or merely contain the toxic bodies precipitated along with them. In the United Kingdom the work of Sidney Martin, in the separation of toxic substances from the bodies of those who have died from certain diseases, is also worthy of mention. Immunity against toxins also became a subject of investigation, and the result was the discovery of the antitoxic action of the serum of animals immunized against tetanus toxin by E. Behring and Kitazato (1890), and by Tizzoni and Cattani.

A similar result was also obtained in the case of diphtheria. The facts with regard to passive immunity were thus established and were put to practical application by the introduction of diphtheria antitoxin as a therapeutic agent in 1894. The technique of serum preparation has become since that time greatly elaborated and improved, the work of P. Ehrlich in this respect being specially noteworthy. The laws of passive immunity were shown to hold also in the case of immunity against living organisms by R. Pfeiffer (1894), and various anti-bacterial sera have been introduced. Of these the anti-streptococcic serum of A. Marmorek (1895) is one of the best known. The principles of protective inoculation have been developed and practically applied on a large scale, notably by W. M. W. Haffkine in the case of cholera (1893) and plague (1896), and more recently by Wright and Semple in the case of typhoid fever. One other discovery of great importance may be mentioned, viz. the agglutinative action of the serum of a patient suffering from a bacterial disease, first described in the case of typhoid fever independently by Widal and by Griinbaum in 1896, though led up to by the work of Pfeiffer, Gruber and Durham and others. Thus a new aid was added to medical science, viz. serum diagnosis of disease. The last decade of the 19th century will stand out in the history of medical science as the period in which serum therapeutics and serum diagnosis had their birth.

In recent years the relations of toxin and antitoxin, still obscure, have been the subject of much study and controversy. It was formerly supposed that the injection of attenuated cultures or dead organisms-vaccines in the widest sensewas only of service in producing immunity as a preventive measure against the corresponding organism, but the work of Sir Almroth Wright has shown that the use of such vaccines may be of service even after infection has occurred, especially when the resulting disease is localized. In this case a general reaction is stimulated by the vaccine which may aid in the destruction of the invading organisms. In regulating the administration of such vaccines he has introduced the method of observing the opsonic index, to which reference is made below. Of the discoveries of new organisms the most important is that of the Spirochaete pallida in syphilis by Schaudinn and Hoffmann in 1905; and although proof that it is the cause of the disease is not absolute, the facts that have been established constitute very strong presumptive evidence in favour of this being the case. It may be noted, however, that it is still doubtful whether this organism is to be placed amongst the bacteria or amongst the protozoa.

The methods employed in studying the relation of bacteria to disease are in principle comparatively simple,but considerable experience and great care are necessary in applying them and in interpreting results. In any given disease there are three chief steps, viz. (i) the discovery of a bacterium in the affected tissues by means of the microscope; (2) the obtaining of the bacterium in pure culture; and (3) the production of the disease by inoculation with a pure culture. By means of microscopic examination more than one organism may sometimes be observed in the tissues,but one single organism by its constant presence and special relations to the tissue changes can usually be selected as the probable cause of the disease, and attempts towards its cultivation can then be made. Such microscopic examination requires the use of the finest lenses and the application of various staining methods. In these latter the basic aniline dyes in solution are almost exclusively used, on account of their special affinity for the bacterial protoplasm. The methods vary much in detail, though in each case the endeavour is to colour the bacteria as deeply, and the tissues as faintly, as possible. Sometimes a simple watery solution of the dye is sufficient, but very often the best result is obtained by increasing the staining power, e.g. by addition of weak alkali, application of heat, &c., and by using some substance which acts as a mordant and tends to fix the stain to the bacteria. Excess of stain is afterwards removed from the tissues by the use of decolorizing agents, such as acids of varying strength and concentration, alcohol, &c. Different bacteria behave very differently to stains; some take them up rapidly, others slowly, some resist decolorization, others are easily decolorized. In some instances the stain can be entirely removed from the tissues, leaving the bacteria alone coloured, and the tissues can then be stained by another colour. This is the case in the methods for staining the tubercle bacillus and also in Gram's method, the essential point in which latter is the treatment with a solution of iodine before decolorizing. In Gram's method, however, only some bacteria retain the stain, while others lose it. The tissues and fluids are treated by various histological methods, but, to speak generally, examination is made either in films smeared on thin cover-glasses and allowed to dry, or in thin sections cut by the microtome after suitable fixation and hardening of the tissue. In the case of any bacterium discovered, observation must be made in a long series of instances in order to determine its invariable presence.

In cultivating bacteria outside the body various media to serve as food material must be prepared and sterilized by heat. The general principle in their preparation is to supply cutt;va- the nutriment for bacterial growth in a form as nearly g y similar as possible to that of the natural habitat of the organisms - in the case of pathogenic bacteria, the natural fluids of the body. The media are used either in a fluid or solid condition, the latter being obtained by a process of coagulation, or by the addition of a gelatinizing agent, and are placed in glass tubes or flasks plugged with cotton-wool. To mention examples, blood serum solidified at a suitable temperature is a highly suitable medium, and various media are made with extract of meat as a basis, with the addition of gelatine or agar as solidifying agents and of non-coagulable proteids (commercial " pep tone ") to make up for proteids lost by coagulation in the preparation. The reaction of the media must in every case be carefully attended to, a neutral or slightly alkaline reaction being, as a rule, most suitable; for delicate work it may be necessary to standardize the reaction by titration methods. The media from the store-flasks are placed in glass test-tubes or small flasks, protected from contamination by cotton-wool plugs, and are sterilized by heat. For most purposes the solid media are to be preferred, since bacterial growth appears as a discrete mass and accidental contamination can be readily recognized. Cultures are made by transferring by means of a sterile platinum wire a little of the material containing the bacteria to the medium. The tubes, after being thus inoculated, are kept at suitable temperatures, usually either at 37° C., the temperature of the body, or at about 20 0 C., a warm summer temperature, until growth appears. For maintaining a constant temperature incubators With regulating apparatus are used. Subsequent cultures or, as they are called, " subcultures," may be made by inoculating fresh tubes, and in this way growth may be maintained often for an indefinite period. The simplest case is that in which only one variety of bacterium is present, and a " pure culture " may then be obtained at once. When, however, several species are present together, means must be adopted for separating them. For this purpose various methods have been devised, the most important being the plate-method of Koch. In this method the bacteria are distributed in a gelatine or agar medium liquefied by heat, and the medium is then poured out on sterile glass plates or in shallow glass dishes, and allowed to solidify. Each bacterium capable of growth gives rise to a colony visible to the naked eye, and if the colonies are sufficiently apart, an inoculation can be made from any one to a tube of culture-medium and a pure culture obtained. Of course, in applying the method means must be adopted for suitably diluting the bacterial mixture. Another important method consists in inoculating an animal with some fluid containing the various bacteria. A pathogenic bacterium present may invade the body, and may be obtained in pure culture from the internal organs. This method applies especially to pathogenic bacteria whose growth on culture media is slow, e.g. the tubercle bacillus.

The full description of a particular bacterium implies an account not only of its microscopical characters, but also of its growth characters in various culture media, its biological properties, and the effects produced in animals by inoculation. To demonstrate readily its action on various substances, certain media have been devised. For example, various sugars - lactose, glucose, saccharose, &c. - are added to test the fermentative action of the bacterium on these substances; litmus is added to show changes in reaction, specially standardized media being used for estimating such changes; peptone solution is commonly employed for testing whether or not the bacterium forms indol; sterilized milk is used as a culture medium to determine whether or not it is curdled by the growth. Sometimes a bacterium can be readily recognized from one or two characters, but not infrequently a whole series of tests must be made before the species is determined. As our knowledge has advanced it has become abundantly evident that the so-called pathogenic bacteria are not organisms with special features, but that each is a member of a group of organisms possessing closely allied characters. From the point of view of evolution we may suppose that certain races of a group of bacteria have gradually acquired the power of invading the tissues of the body and producing disease. In the acquisition of pathogenic properties some of their original characters have become changed, but in many instances this has taken place only to a slight degree, and, furthermore, some of these changes are not of a permanent character. It is to be noted that in the case of bacteria we can only judge of organisms being of different species by the stability of the characters which distinguish them, and numerous examples might be given where their characters become modified by comparatively slight change in their environment. The cultural as well as the microscopical characters of a pathogenic organism may be closely similar to other non-pathogenic members of the same group, and it thus comes to be a matter of extreme difficulty in certain cases to state what criterion should be used in differentiating varieties. The tests which are applied for this purpose at present are chiefly of two kinds. In the first place, such organisms may be differentiated by the chemical change produced by them in various culture media, e.g. by their fermentative action on various sugars, &c., though in this case such properties may become modified in the course of time. And in the second place, the various serum reactions to be described below have been called into requisition. It may be stated that the introduction of a particular bacterium into the tissues of the body leads to certain properties appearing in the serum, which are chiefly exerted towards this particular bacterium. Such a serum may accordingly within certain limits be used for differentiating this organism from others closely allied to it (vide infra). The modes of cultivation described apply only to organisms which grow in presence of oxygen. Some, however - the strictly anaerobic bacteria - grow only in the absence of oxygen; hence means must be adopted for excluding this gas. It is found that if the inoculation be made deep down in a solid medium, growth of an anaerobic organism will take place, especially if the medium contains some reducing agent such as glucose. Such cultures are called " deep cultures." To obtain growth of an anaerobic organism on the surface of a medium, in using the plate method, and also for cultures in fluids, the air is displaced by an indifferent gas, usually hydrogen.

In testing the effects of bacteria by inoculation the smaller rodents, rabbits, guinea-pigs, and mice, are usually employed. One great drawback in certain cases is that such animals are not susceptible to a given bacterium, or that the disease is different in character from that in the human subject. In some cases, e.g. Maltafeverand relapsing fever, monkeys have been used with success, but in others, e.g. leprosy, none of the lower animals has been found to be susceptible. Discretion must therefore be exercised in interpreting negative results in the lower animals. For purposes of inoculation young vigorous cultures must be used. The bacteria are mixed with some indifferent fluid, or a fluid culture is employed. The injections are made by means of a hypodermic syringe into the subcutaneous tissue, into a vein, into one of the serous sacs, or more rarely into some special part of the body. The animal, after injection, must be kept in favourable surroundings, and any resulting symptoms noted. It may die, or may be killed at any time desired, and then a post-mortem examination is made, the conditions of the organs, &c., being observed and noted. The various tissues affected are examined microscopically and cultures made from them; in this way the structural changes and the relation of bacteria to them can be determined.

Though the causal relationship of a bacterium to a disease may be completely established by the methods given, another very important part of bacteriology is concerned with the poisons or toxins formed by bacteria. These toxins may become free in the culture fluid, and the living bacteria may then be got rid of by filtering the fluid through a filter of unglazed porcelain, whose pores are sufficiently small to retain them. The passage of the fluid is readily effected by negative pressure produced by an ordinary water exhaust-pump.The P Y Y effects of the filtrate are then tested by the methods used in pharmacology. In other instances the toxins are retained to a large extent within the bacteria, and in this case the dead bacteria are injected as a suspension in fluid. Methods have been introduced for the purpose of breaking up the bodies of bacteria and setting free the intracellular toxins. For this purpose Koch ground up tubercle bacilli in an agate mortar and treated them with distilled water until practically no deposit remained. Rowland and Macfadyen for the same purpose introduced the method of grinding the bacilli in liquid air. At this temperature the bacterial bodies are extremely brittle, and are thus readily broken up. The study of the nature of toxins requires, of course, the various methods of organic chemistry. Attempts to obtain them in an absolutely pure condition have, however, failed in important cases. So that when a " toxin " is spoken of, a mixture with other organic substances is usually implied. Or the toxin may be precipitated with other organic substances, purified to a certain extent byre-solution, re-precipitation, &c., and desiccated. A " dry toxin " is thus obtained, though still in an impure condition. Toxic substances have also been separated by corresponding methods from the bodies of those who have died of certain diseases, and the action of such substances on animals is in some cases an important point in the pathology of the disease. Another auxiliary method has been applied in this department, viz. the separation of organic substances by filtration under high pressure through a colloid membrane, gelatine supported in the pores of a porcelain filter being usually employed. It has been found, for example, that a toxin may pass through such a filter while an antitoxin may not. The methods of producing immunity are dealt with below.

The fact that in anthrax, one of the first diseases to be fully studied, numerous bacilli are present in the blood of infected animals, gave origin to the idea that the organisms might produce their effect by using up the oxygen g ' P Y g P Yg of the blood. Such action is now known to be quite a of subsidiary matter. And although effects may some times be produced in a mechanical manner by bacteria plugging capillaries of important organs, e.g. brain and kidneys, it may now be stated as an accepted fact that all the important results of bacteria in the tissues are due to poisonous bodies or toxins formed by them. Here, just as in the general subject of fermentation, we must inquire whether the bacteria form the substances in question directly or by means of non-living ferments or enzymes. With regard to toxin formation the following general statements may be made. In certain instances, e.g. in the case of the tetanus and diphtheria bacilli, the production of soluble toxins can be readily demonstrated by filtering a culture in bouillon germ-free by means of a porcelain filter, and then injecting some of the filtrate into an animal. In this way the characteristic features of the disease can be reproduced. Such toxins being set free in the culture medium are often known as extracellular. In many cases, however, the filtrate, when injected, produces comparatively little effect, whilst toxic action is observed when the bacteria in a dead condition are used; this is the case with the organisms of tubercle, cholera, typhoid and many others. The toxins are here manifestly contained within the bodies of the bacteria, i.e. are intracellular, though they may become free on disintegration of the bacteria. The action of these intracellular toxins has in many instances nothing characteristic, but is merely in the direction of producing fever and interfering with the vital processes of the body generally, these disturbances often going on to a fatal result. In other words, the toxins of different bacteria are closely similar in their results on the body and the features of the corresponding diseases are largely regulated by the vital properties of the bacteria, their distribution in the tissues, &c. The distinction between the two varieties of toxins, though convenient. must not be pushed too far, as we know little regarding their mode of formation. Although the formation of toxins with characteristic action can be shown by the above methods, yet in some cases little or no toxic action can be demonstrated. This, for example, is the case with the anthrax bacillus; although the effect of this organism in the living body indicates the production of toxins which diffuse for a distance around the bacteria. This and similar facts have suggested that some toxins are only produced in the living body.

A considerable amount of work has been done in connexion with this subject, and many observers have found that fluids taken from the living body in which the organisms have been growing, contain toxic substances, to which the name of aggressins has been applied. Fluid containing these aggressins greatly increases the toxic effect of the corresponding bacteria, and may produce death at an earlier stage than ever occurs with the bacteria alone. They also appear to have in certain cases a paralysing action on the cells which act as phagocytes. The work on this subject is highly suggestive, and opens up new possibilities with regard to the investigation of bacterial action within the body. Not only are the general symptoms of poisoning in bacterial disease due to toxic substances, but also the tissue changes, many of them of inflammatory nature, in the neighbourhood of the bacteria. Thus, to mention examples, diphtheria toxin produces inflammatory oedema which may be followed by necrosis; dead tubercle bacilli give rise to a tubercle-like nodule, &c. Furthermore, a bacillus may give rise to more than one toxic body, either as stages in one process of change or as distinct products. Thus paralysis following diphtheria is in all probability due to a different toxin from that which causes the acute symptoms of poisoning or possibly to a modification of it sometimes formed in specially large amount. It is interesting to note that in the case of the closely analogous example of snake venoms, there may be separated from a single venom a number of toxic bodies which have a selective action on different animal tissues.

Regarding the chemical nature of toxins less is known than regarding their physiological action. Though an enormous of amount of work has been done on the subject, no important bacterial toxin has as yet been obtained in a pure condition, and, though many of them are probably of proteid nature, even this cannot be asserted with absolute certainty. Brieger, in his earlier work, found that alkaloids were formed by bacteria in a variety of conditions, and that some of them were poisonous. These alkaloids he called ptomaines. The methods used in the investigations were, however, open to objection, and it is now recognized that although organic bases may sometimes be formed, and may be toxic, the important toxins are not of that nature. A later research by Brieger along with Fraenkel pointed to the extracellular toxins of diphtheria, tetanus and other diseases being of proteid nature, and various other observers have arrived at a like conclusion. The general result of such research has been to show that the toxic bodies are, like proteids, precipitable by alcohol and various salts; they are soluble in water, are somewhat easily dialysable, and are relatively unstable both to light and heat. Attempts to get a pure toxin by repeated precipitation and solution have resulted in the production of a whitish amorphous powder with highly toxic properties. Such a powder gives a proteid reaction, and is no doubt largely composed of albumoses, hence the name toxalbumoses has been applied. The question has, however, been raised whether the toxin is really itself a proteid, or whether it is not merely carried down with the precipitate. Brieger and Boer, by precipitation with certain salts, notably of zinc, obtained a body which was toxic but gave no reaction of any form of proteid. There is of course the possibility in this case that the toxin was a proteid, but was in so small amount that it escaped detection. These facts show the great difficulty of the problem, which is probably insoluble by present methods of analysis; the only test, in fact, for the existence of a toxin is its physiological effect. It may also be mentioned that many toxins have now been obtained by growing the particular organism in a proteid-free medium, a fact which shows that if the toxin is a proteid it may be formed synthetically by the bacterium as well as by modification of proteid already present. With regard to the nature of intracellular toxins, there is even greater difficulty in the investigation and still less is known. Many of them, probably also of proteid nature, are much more resistant to heat; thus the intracellular toxins of the tubercle bacillus retain certain of their effects even after exposure to ioo° C. Like the extracellular toxins they may be of remarkable potency; for example, fever is produced in the human subject by the injection into the blood of an extremely minute quantity of dead typhoid bacilli. We cannot as yet speak definitely with regard to the part played by enzymes in these toxic processes. Certain toxins resemble enzymes as regards their conditions of precipitation and relative instability, and the fact that in most cases a considerable period intervenes between the time of injection and the occurrence of symptoms has been adduced in support of the view that enzymes are present. In the case of diphtheria Sidney Martin obtained toxic albumoses in the spleen, which he considered were due to the digestive action of an enzyme formed by the bacillus in the membrane and absorbed into the circulation. According to this view, then, a part at least of the directly toxic substance is produced in the living body by enzymes present in the so-called toxin obtained from the bacterial culture. Recent researches go to show that enzymes play a greater part in fermentation by living ferments than was formerly supposed, and by analogy it is likely that they are also concerned in the processes of disease. But this has not been proved, and hitherto no enzyme has been separated from a pathogenic bacterium capable of forming, by digestive or other action, the toxic bodies from proteids outside the body. It is also to be noted that, as in the case of poisons of known constitution, each toxin has a minimum lethal dose which is proportionate to the weight of the animal and which can be ascertained with a fair degree of accuracy.

The action of toxins is little understood. It consists in all probability of disturbance, by means of the chemical affinities of the toxin, of the highly complicated molecules of living cells. This disturbance results in disintegration to a varying degree, and may produce changes visible on microscopic examination. In other cases such changes cannot be detected, and the only evidence of their occurrence may be the associated symptoms. The very important work of Ehrlich on diphtheria toxin shows that in the molecule of toxin there are at least two chief atom groups - one, the " haptophorous," by which the toxin molecule is attached to the cell protoplasm; and the other the " toxophorous," which has a ferment-like action on the living molecule, producing a disturbance which results in the toxic symptoms. On this theory, susceptibility to a toxin will imply both a chemical affinity of certain tissues for the toxin molecule and also sensitiveness to its actions, and, furthermore, non-susceptibility may result from the absence of either of these two properties.

A bacterial infection when analysed is seen to be of the nature of an intoxication. There is, however, another all-important factor concerned, viz. the multiplication of the living organisms in the tissues; this is essential to, and regulates, the supply of toxins. It is important that these two essential factors should be kept clearly in view, since the means of defence against any disease may depend upon the power either of neutralizing toxins or of killing the organisms producing them. It is to be noted that there is no fixed relation between toxin production and bacterial multiplication in the body, some of the organisms most active as toxin producers having comparatively little power of invading the tissues.

We shall now consider how bacteria may behave when they have gained entrance to the body, what effects may be produced, and what circumstances may modify the disease in any particular case. The extreme instance of bacterial invasion is found in some of the septicaemias in the lower animals, e.g. anthrax septicaemia in guinea-pigs, pneumococcus septicaemia in rabbits. In such diseases the bacteria, when introduced into the subcutaneous tissue, rapidly gain entrance to the blood stream and multiply freely in it, and by means of their toxins cause symptoms of general poisoning.

A widespread toxic action is indicated by the lesions found - cloudy swelling, which may be followed by fatty degeneration, in internal organs, capillary haemorrhages, &c. In septicaemia. in the human subject, often due to streptococci, the process is similar, but the organisms are found especially in the capillaries of the internal organs and may not be detectable in the peripheral circulation during life. In another class of diseases, the organisms. first produce some well-marked local lesion, from which secondary extension takes place by the lymph or blood stream to other parts of the body, where corresponding lesions are formed. In this way secondary abscesses, secondary tubercle glanders and nodules, &c., result; in typhoid fever there is secondary invasion of the mesenteric glands, and clumps of bacilli are also found in internal organs, especially the spleen, though there may be little tissue change around them. In all such cases there is seen a selective character in the distribution of the lesions, some organs being in any disease much more liable to infection than others. .In still another class of diseases the bacteria are restricted to some particular part of the body, and the symptoms are due to toxins which are absorbed from it. Thus in cholera the bacteria are practically confined to the intestine, in diphtheria to the region of the false membrane, in tetanus to some wound. In the lastmentioned disease even the local multiplication depends upon the presence of other bacteria, as the tetanus bacillus has practically no power of multiplying in the healthy tissues when introduced alone.

The effects produced by bacteria may be considered under the following heads: (I) tissue changes produced in the vicinity of the bacteria, either at the primary or secondary foci; (2) tissue changes produced at a distance by absorption of their toxins; (3) symptoms. The changes in the vicinity of bacteria are to be regarded partly as the direct result of the action of toxins on living cells, and partly as indicating a reaction on the part of the tissues. (Many such changes are usually grouped together under the heading of " inflammation " of varying degree--acute, subacute and chronic.) Degeneration and death of cells, haemorrhages, serous and fibrinous exudations, leucocyte emigration, proliferation of connective tissue and other cells, may be mentioned as some of the fundamental changes. Acute inflammation of various types, suppuration, granulation-tissue formation, &c., represent some of the complex resulting processes. The changes produced at a distance by distribution of toxins may be very manifold - cloudy swelling and fatty degeneration, serous effusions, capillary haemorrhages, various degenerations of muscle, hyaline degeneration of small blood-vessels, and, in certain chronic diseases, waxy degeneration, all of which may be widespread, are examples of the effects of toxins, rapid or slow in action. Again, in certain cases the toxin has a special affinity for certain tissues. Thus in diphtheria changes in both nerve cells and nerve fibres have been found, and in tetanus minute alterations in the nucleus and protoplasm of nerve cells.

The lesions mentioned are in many instances necessarily accompanied by functional disturbances or clinical symptoms, symptoms. varying according to site, and to the nature and degree of the affection. In addition, however, there occur in bacterial diseases symptoms to which the correlated structural changes have not yet been demonstrated. Amongst these, the most important is fever with increased protein metabolism, attended with disturbances of the circulatory and respiratory systems. Nervous symptoms, somnolence, coma, spasms, convulsions and paralysis are of common occurrence. All such phenomena, however, are likewise due to the disturbance of the molecular constitution of living cells. Alterations in metabolism are found to be associated with some of these, but with others no corresponding physical change can be demonstrated. The action of toxins on various glands, producing diminished or increased functional activity, has a close analogy to that of certain drugs. In short, if we place aside the outstanding exception of tumour growth, we may say that practically all the important phenomena met with in disease may be experimentally produced by the injection of bacteria or of their toxins.

The result of the entrance of a virulent bacterium into the tissues of an animal is not a disease with hard and fast characters, but varies greatly with circumstances. With regard to the subject of infection the chief factor is susceptibility; with regard to the bacterium virulence is allimportant. Susceptibility, as is well recognized, varies much under natural conditions in different species, in different races of the same species, and amongst individuals of the same race. It also varies with the period of life, young subjects being more susceptible to certain diseases, e.g. diphtheria, than adults. Further, there is the very important factor of acquired susceptibility. It has been experimentally shown that conditions such as fatigue, starvation, exposure to cold, &c., lower the general resisting powers and increase the susceptibility to bacterial infection. So also the local powers of resistance may be lowered by injury or depressed vitality. In this way conditions formerly believed to be the causes of disease are now recognized as playing their part in predisposing to the action of the true causal agent, viz. the bacterium. In health the blood and internal tissues are bacterium-free; after death they offer a most suitable pabulum for various bacteria; but between these two extremes lie states of varying liability to infection. It is also probable that in a state of health organisms do gain entrance to the blood from time to time and are rapidly killed off. The circumstances which alter the virulence of bacteria will be referred to again in connexion with immunity, but it may be stated here that, as a general rule, the virulence of an organism towards an animal is increased by sojourn in the tissues of that animal. The increase of virulence becomes especially marked when the organism is inoculated from animal to animal in series, the method of passage. This is chiefly to be regarded as an adaptation to surroundings, though the fact that the less virulent members of the bacterial species will be liable to be killed off also plays a part.;,Conversely, the virulence tends to diminish on cultivation on artificial media outside the body, especially in circumstances little favourable to growth.

By immunity is meant non-susceptibility to a given disease, or to experimental inoculation with a given bacterium or toxin. The term must be used in a relative sense, and account must always be taken of the conditions present. An immunity. animal may be readily susceptible to a disease on experimental inoculation, and yet rarely or never suffer from it naturally, because the necessary conditions of infection are not supplied in nature. That an animal possesses natural immunity can only be shown on exposing it to such conditions, this being usually most satisfactorily done in direct experiment. Further, there are various degrees of immunity, and in this connexion conditions of local or general diminished vitality play an important part in increasing the susceptibility. Animals naturally susceptible may acquire immunity, on the one hand by successfully passing through an attack of the disease, or, on the other hand, by various methods of inoculation. Two chief varieties of artificial immunity are now generally recognized, differing chiefly according to the mode of production. In the first - active immunity - a reaction or series of reactions is produced in the body of the animal, usually by injections of bacteria or their products. The second - passive immunity - is produced by the transference of a quantity of the serum of an animal actively immunized to a fresh animal; the term is applied because there is brought into play no active change in the tissues of the second animal. The methods of active immunity have been practically applied in preventive inoculation against disease; those of passive immunity have given us serum therapeutics. The chief facts with regard to each may now be stated.

r. Active Immunity

The key to the artificial establishment of active immunity is given by the fact long established that recovery from an attack of certain infective diseases is accompanied by protection for varying periods of time against a subsequent attack. Hence follows the idea of producing a modified attack of the disease as a means of prevention - a principle which had been previously applied in inoculation against smallpox. Immunity, however, probably results from certain substances introduced into the system during the disease rather than from the disease itself; for by properly adjusted doses of the poison (in the widest sense), immunity may result without any symptoms of the disease occurring. Of the chief methods used in producing active immunity the first is by inoculation with bacteria whose virulence has been diminished, i.e. with an " attenuated virus." Many of the earlier methods of attenuation were devised in the case of the anthrax bacillus, an organism which is, however, somewhat exceptional as regards the relative stability of its virulence. Many such methods consist, to speak generally, in growing the organism outside the body under somewhat unsuitable conditions, e.g. at higher temperatures than the optimum, in the presence of weak antiseptics, &c. The virulence of many organisms, however, becomes diminished when they are grown on the ordinary artificial media, and the diminution is sometimes accelerated by passing a current bility. of air over the surface of the growth. Sometimes also the virulence of a bacterium for a particular kind of animal becomes lessened on passing it through the body of one of another species. Cultures of varying degree of virulence may be obtained by such methods, and immunity can be gradually increased by inoculation with vaccines of increasing virulence. The immunity may be made to reach a very high degree by ultimately using cultures of intensified virulence, this " supervirulent " character being usually attained by the method of passage already explained.

A second method is by injection of the bacterium in the dead condition, whereby immunity against the living organism may be produced. Here manifestly the dose may be easily controlled, and may be gradually increased in successive inoculations. This method has a wide application. A third method is by injections of the separated toxins of a bacterium, the resulting immunity being not only against the toxin, but, so far as present knowledge shows, also against the living organism. In the development of toxin-immunity the doses, small at first, are gradually increased in successive inoculations; or, as in the case of very active toxins, the initial injections are made with toxin modified by heat or by the addition of various chemical substances. Immunity of the same nature can be acquired in the same way against snake and scorpion poisons, and against certain vegetable toxins, e.g. ricin, abrin, &c.

In order that the immunity may reach a high degree, either the bacterium in a very virulent state or a large dose of toxin must ultimately be used in the injections. In such cases the immunity is, to speak generally, specific, i.e. applies only to the bacterium or toxin used in its production. A certain degree of non-specific immunity or increased tissue resistance may be produced locally, e.g. in the peritoneum, by injections of nonpathogenic organisms, peptone, nucleic acid and various other substances. In these cases the immunity is without specific character, and cannot be transferred to another animal. Lastly, in a few instances one organism has an antagonistic action to another; for example, the products of B. pyocyaneus have a certain protective action against B. anthracis. This method has, however, not yielded any important practical application.

2. Passive Immunity: Anti-sera. - The development of active immunity by the above methods is essentially the result of a reactive process on the part of the cells of the body, though as yet we know little of its real nature. It is, however, also accompanied by the appearance of certain bodies in the blood serum of the animal treated, to which the name of anti-substances is given, and these have been the subject of extensive study. It is by means of them that immunity (passive) can be transferred to a fresh animal. The development of anti-substances is, however, not peculiar to bacteria, but occurs also when alien cells of various kinds, proteins, ferments, &c., are injected. In fact, organic molecules can be divided into two classes according as they give rise to anti-substances or fail to do so. Amongst the latter, the vegetable poisons of known constitution, alkaloids, glucosides, &c., are to be placed. The molecules which lead to the production of anti-substances are usually known as antigens, and each antigen has a specific combining affinity for its corresponding anti-substance, fitting it as a lock does a key. The antigens, as already indicated, may occur in bacteria, cells, &c., or they may occur free in a fluid. Anti-substances may be arranged, as has been done by Ehrlich, into three main groups. In the first group, the anti-substance simply combines with the antigen, without, so far as we know, producing any change in it. The antitoxins are examples of this variety. In the second group, the anti-substance, in addition to combining with the antigen, produces some recognizable physical change in it; the precipitins and agglutinins may be mentioned as examples. In the third group, the anti-substance, after it has combined with the antigen, leads to the union of a third body called complement (alexine or cytase of French writers), which is present in normal serum. As a result of the union of the three substances, a dissolving or digestive action is often to be observed. This is the mode of action of the anti-substances in the case of a haemolytic or bacteriolytic serum. So far as bacterial immunity is concerned, the anti-serum exerts its action either on the toxin or on the bacterium itself; that is, its action is either antitoxic or anti-bacterial. The properties of these two kinds of serum may now be considered.

The term " antitoxic " signifies that serum has the power of neutralizing the action of the toxin, as is shown by mixing them together outside the body and then injecting them into an animal. The antitoxic serum when injected enttinoxic previously to the toxin also confers immunity (passive) against it; when injected after the toxin it has within certain limits a curative action, though in this case its dose requires to be large. The antitoxic property is developed in a susceptible animal by successive and gradually increasing doses of the toxin. In the earlier experiments on smaller animals the potency of the toxin was modified for the first injections, but in preparing antitoxin for therapeutical purposes the toxin is used in its unaltered condition, the horse being the animal usually employed. The injections are made subcutaneously and afterwards intravenously; and, while the dose must be gradually increased, care must be taken that this is not done too quickly, otherwise the antitoxic power of the serum may fall and the health of the animal suffer. The serum of the animal is tested from time to time against a known amount of toxin, i.e. is standardized. The unit of antitoxin in Ehrlich's new standard is the amount requisite to antagonize i oo times the minimum lethal dose of a particular toxin to a guinea-pig of 250 grm. weight, the indication that the toxin has been antagonized being that a fatal result does not follow within five days after the injection. In the case of diphtheria the antitoxic power of the serum may reach Boo units per cubic centimetre, or even more. The laws of antitoxin production and action are not confined to bacterial toxins, but apply also to other vegetable and animal toxins, resembling them in constitution, viz. the vegetable toxalbumoses and the snake-venom group referred to above.

The production of antitoxin is one of the most striking facts of biological science, and two important questions with regard to it must next be considered, viz. how does the antitoxin Action of act ? and how is it formed within the body ? Theo- antitoxin. retically there are two possible modes of action: antitoxin may act by means of the cells of the body, i.e. indirectly or physiologically; or it may act directly on the toxin, i.e. chemically or physically. The second view may now be said to be established, and, though the question cannot be fully discussed here, the chief grounds in support of a direct action may be given. (a) The action of antitoxin on toxin, as tested by neutralization effects, takes place more quickly in concentrated than in weak solutions, and more quickly at a warm (within certain limits) than at a cold temperature. (b) Antitoxin acts more powerfully when injected along with the toxin than when injected at the same time in another part of the body; if its action were on the tissue-cells one would expect that the site of injection would be immaterial. For example, the amount necessary to neutralize five times the lethal dose being determined, twenty times that amount will neutralize a hundred times the lethal dose. In the case of physiological antagonism of drugs this relationship does not hold. (c) It has been shown by C. J. Martin and Cherry, and by A. A. Kanthack and Cobbett, that in certain instances the toxin can be made to pass through a gelatine membrane, whereas the antitoxin cannot, its molecules being of larger size. If, however, toxin be mixed with antitoxin for some time, it can no longer be passed through, presumably because it has become combined with the antitoxin.

Lastly it may be mentioned that when a toxin has some action which can be demonstrated in a test-tube experiment, for example, a dissolving action on red corpuscles, this action may be annulled by previously adding the antitoxin to toxin; in such a case the intervention of the living tissues is excluded. In view of the fact that antitoxin has a direct action on toxin, we may say that theoretically this may take place in one of two ways. It may produce a disintegration of the toxin molecule, or it may combine with it to produce a body whose combining affinities are satisfied. The latter view, first advocated by Ehrlich, harmonizes with the facts established with regard to toxic action and the behaviour of antitoxins, and may now be regarded as established. His view as to the dual composition of the toxin molecule has already been mentioned, and it is evident that if the haptophorous or combining group has its affinity satisfied by union with antitoxin, the toxin will no longer combine with living cells, and will thus be rendered harmless. One other important fact in support of what has been stated is that a toxin may have its toxic action diminished, and may still require the same amount of antitoxin as previously for neutralization. This is readily intelligible on the supposition that the toxophorous group is more labile than the haptophorous. There is, however,, still dispute with regard to the exact nature of the union of toxin and antitoxin. Ehrlich's view is that the two substances form a firm combination like a strong acid and a base. He found, however, that if he took the largest amount of toxin which was just neutralized by a given amount of antitoxin, much more than a single dose of toxin had to be added before a single dose was left free. For example, if 'co doses of toxin were neutralized by a unit of antitoxin (v. supra) it might be that 12 5 doses would need to be added to the same amount of antitoxin before the mixture produced a fatal result when it was injected. This result, which is usually known now as the " Ehrlich phenomenon," was explained by him on the supposition that the " toxin " does not represent molecules which are all the same, but contains molecules of different degrees of combining affinity and of toxic action. Accordingly, the most actively toxic molecules will be neutralized first, and those which are left over, that is, uncombined with antitoxin, will have a weaker toxic action. This view has been assailed by Thorvald Madsen and S. A. Arrhenius, who hold that the union of toxin and antitoxin is comparatively loose, and belongs to the classof reversible actions, being comparable in fact with the union of a weak acid and base. If such were the condition there would always be a certain amount both of free toxin and of free antitoxin in the mixture, and in this case also considerably more than a dose of toxin would have to be added to a " neutral mixture " before the amount of free toxin was increased by a dose, that is, before the mixture became lethal. It may be stated that while in certain instances the union of toxin and antitoxin may be reversible, all the facts established cannot be explained on this simple hypothesis of reversible action. Still another view, advocated by Bordet, is that the union of toxin and antitoxin is rather of physical than of strictly chemical nature, and represents an interaction of colloidal substances, a sort of molecular deposition by which the smaller toxin molecule becomes entangled in the larger molecule of antitoxin. Sufficient has been said to show that the subject is one of great intricacy, and no simple statement with regard to it is as yet possible. We are probably safe in saying, however, that the molecules of a toxin are not identical but vary in the degree of their combining affinities, and also in their toxic action, and that, while in some cases the combination of anti-substances has been shown to be reversible, we are far from being able to say that this is a general law.

The origin of antitoxin is of course merely a part of the general question regarding the production of anti-substances in general, as these all combine in the same way with their homo logous substances and have the same character of g toxin. specificity. As, however, most of the work has been done with regard to antitoxin production we may consider here the theoretical aspect of the subject. There are three chief possibilities: (a) that the antitoxin is a modification of the toxin; (b) that it is a substance normally present, but produced in excess under stimulation of the toxin; (c) that it is an entirely new product. The first of these, which would imply a process of a very remarkable nature, is disproved by what is observed after bleeding an animal whose blood contains antitoxin. In such a case it has been shown that, without the introduction of fresh toxin, new antitoxin appears, and therefore must be produced by the living tissues. The second theory is the more probable a priori, and if established removes the necessity for the third. It is strongly supported by Ehrlich, who, in his so-called " side-chain " (Seitenkette) theory, explains antitoxin production as an instance of regeneration after loss. Living protoplasm, or in other words a biogen molecule, is regarded as consisting of a central atom group (Leistungskern), related to which are numerous secondary atom groups or sidechains, with unsatisfied chemical affinities. The' side-chains constitute the means by which other molecules are added to the living molecule, e.g. in the process of nutrition. It is by means of such side-chains that toxin molecules are attached to the protoplasm, so that the living molecules are brought under the action of the toxophorous groups of the toxins. In antitoxin production this combination takes place, though not in sufficient amount to produce serious toxic symptoms. It is further supposed that the combination being of somewhat firm character, the side-chains thus combined are lost for the purposes of the cell and are therefore thrown off. By the introduction of fresh toxin the process is repeated and the regeneration of side-chains is increased. Ultimately the regeneration becomes an over-regeneration and free side-chains produced in excess are set free and appear in the blood as antitoxin molecules. In other words the substances, which when forming part of the cells fix the toxin to the cells, constitute antitoxin molecules when free in the serum. This theory, though not yet established, certainly affords the most satisfactory explanation at present available. In support of it there is the remarkable fact, discovered by A. Wassermann and Takaki in the case of tetanus, that there do exist in the nervous system molecules with combining affinity for the tetanus toxin. If, for example, the brain and spinal cord removed from an animal be bruised and brought into contact with tetanus toxin, a certain amount of the toxicity disappears, as shown by injecting the mixture into another animal. Further, these molecules in the nervous system present the same susceptibility to heat and other physical agencies as does tetanus antitoxin. There is therefore strong evidence that antitoxin molecules do exist as part of the living substance of nerve cells. It has, moreover, been found that the serum of various animals has a certain amount of antitoxic action, and thus the basis for antitoxin production, according to Ehrlich's theory, is afforded. The theory also supplies the explanation of the power which an animal possesses of producing various antitoxins, since this depends ultimately upon susceptibility to toxic action. The explanation is thus carried back to the complicated constitution of biogen molecules in various living cells of the body. It may be added that in the case of all the other kinds of anti-substances, which are produced by a corresponding reaction, we have examples of the existence of traces of them in the blood serum under normal conditions. We are, accordingly, justified in definitely concluding that their appearance in large amount in the blood, as the result of active immunization, represents an increased production of molecules which are already present in the body, either in a free condition in its fluids or as constituent elements of its cells.

In preparing anti-bacterial sera the lines of procedure correspond to those followed in the case of antitoxins, but the bacteria themselves in the living or dead condition or their maceration products are always used in the injections. Sometimes dead bacteria, living virulent bacteria, and living supervirulent bacteria, are used in succession, the object being to arrive ultimately at a high dosage, though the details vary in different instances. The serum of an animal thus actively immunized has powerful protective properties towards another animal, the amount necessary for protection being sometimes almost inconceivably small. As a rule it has no action on the corresponding toxin, i.e. is. not antitoxic. In addition to the protective action, such a serum may possess activities which can be demonstrated_ outside the body. Of these the most important are (a) bacteriolytic or lysogenic action, (b) agglutinative action, and (c) opsonic action.

The first of these, lysogenic or bacteriolytic action, consists in.

" the production of a change in the corresponding bacterium whereby it becomes granular, swells up and ultimately may undergo dissolution. Pfeiffer was the first to show that this occurred when the bacterium was injected into the peritoneal cavity of the animal immunized against it, and also when a little of the serum of such an animal was injected with the bacterium into the peritoneum of a fresh, i.e. non-immunized animal. Metchnikoff and Bordet subsequently devised means by which a similar change could be produced in vitro, and analysed the conditions necessary for its occurrence. It has been completely established that in this phenomenon of lysogenesis there are two substances concerned, one specially developed or developed in excess, and the other present in normal serum. The former (Immunko y per of Ehrlich, substance sensibilisatrice of Bordet) is the more stable, resisting a temperature of 60° C., and though giving the specific character to the reaction cannot act alone. The latter is fermentlike and much more labile than the former, being readily destroyed at 60° C. It may be added that the protective power is not lost by exposure to the temperature mentioned, this apparently depending upon a specific anti-substance. Furthermore, lysogenic action is not confined to the case of bacteria but obtains also with other organized structures, e.g. red corpuscles (Bordet, Ehrlich and Morgenroth), leucocytes and spermatozoa (Metchnikoff). That. is to say, if an animal be treated with injections of these bodies, its serum acquires the power of dissolving or of producing some disintegrative effect in them. The development of the immune body with specific combining affinity thus presents an analogy to antitoxin production, the difference being that in lysogenesis another substance is necessary to complete the process. It can be shown that in many cases when bacteria are injected the serum of the treated animal has no bacteriolytic effect, and still an immune body is present, which leads to the fixation of complement; in this case bacteriolysis does not occur, because the organism is not susceptible to the action of the complement. In all cases the important action is the binding of complement to the bacterium by means of the corresponding immune body; whether or not death of the bacterium occurs, will depend upon its susceptibility to the action of the particular complement, the latter acting like a toxin or digestive ferment. It is to be noted that in the process of immunization complement does not increase in amount; accordingly the immune serum comes to contain immune body much in excess of the amount of complement necessary to complete its action. An important point with regard to the therapeutic application of an anti-bacterial serum, is that when the serum is kept in vitro the complement rapidly disappears, and accordingly the complement necessary for the production of the bactericidal action must be supplied by the blood of the patient treated. This latter complement may not suit the immune body, that is, may not be fixed to the bacterium by means of it, or if the latter event does occur, may fail to bring about the death of the bacteria. These circumstances serve, in part at least, to explain the fact that the success attending the use of anti-bacterial sera has been much inferior to that in the case of antitoxic sera.

Another property which may be possessed by an anti-bacterial serum is that of agglutination. By this is meant the aggregation into clumps of the bacteria uniformly distributed (natiai n an indifferent fluid; if the bacterium is motile its movement is arrested during the process. The process is of course observed by means of the microscope, but the clumps soon settle in the fluid and ultimately form a sediment, leaving the upper part clear. This change, visible to the naked eye, is called sedimentation. B. A. Charrin and G. E. H. Roger first showed in the case of B. pyocyaneus that when a small quantity of the homologous serum (i.e. the serum of an animal immunized against the bacterium) was added to a fluid culture of this bacillus, growth formed a sediment instead of a uniform turbidity. Gruber and Durham showed that sedimentation occurred when a small quantity of the homologous serum was added to an emul:_on of the bacterium in a small test-tube, and found that this obtained in all cases where Pfeiffer's lysogenic action could be demonstrated. Shortly afterwards Widal and also Griinbaum showed that the serum of patients suffering from typhoid fever, even at an early stage of the disease, agglutinated the typhoid bacillus - a fact which laid the foundation of serum diagnosis. A similar phenomenon has been demonstrated in the case of Malta fever, cholera, plague, infection with B. coli, " meat-poisoning " due to Gartner's bacillus, and various other infections. As regards the mode of action of agglutinins, Gruber and Durham considered that it consists in a change in the envelopes of the bacteria, by which they swell up and become adhesive. The view has various facts in its support, but F. Kruse and C. Nicolle have found that if a bacterial culture be filtered germ-free, an agglutinating serum still produces some change in it, so that particles suspended in it become gathered into clumps. E. Duclaux, for this reason, considers that agglutinins are coagulative ferments.

The phenomenon of agglutination depends essentially on the union of molecules in the bacteria - the agglutinogens - with the corresponding agglutinins, but another essential is the presence of a certain amount of salts in the fluid, as it can be shown that when agglutinated masses of bacteria are washed salt-free the clumps become resolved. The fact that agglutinins appear in the body at an early stage in a disease has been taken by some observers as indicating that they have nothing to do with immunity, their development being spoken of as a reaction of infection. This conclusion is not justified, as we must suppose that the process of immunization begins to be developed at an early period in the disease, that it gradually increases, and ultimately results in cure. It should also be stated that agglutinins are used up in the process of agglutination, apparently combining with some element of the bacterial structure. In view of all the facts it must be admitted that the agglutinins and immune bodies are the result of corresponding reactive processes, and are probably related to one another. The development of all antagonistic substances which confer the special character on antimicrobic sera, as well as antitoxins, may be expressed as the formation of bodies with specific combining affinity for the organic substance introduced into the system - toxin, bacterium, red corpuscle, &c., as the case may be. The bacterium, being a complex organic substance, may thus give rise to more than one antagonistic or combining substance.

By opsonic action is meant the effect which a serum has on bacteria in making them more susceptible to phagocytosis by the white corpuscles of the blood. Such an effect may be demonstrated outside the body by making a (actiopsonic suitable mixture of (a) a suspension of the particular bacterium, (b) the serum to be tested, and (c) leucocytes of a normal animal or person. The mixture is placed in a thin capillary tube and incubated at 37° C. for half an hour; a film preparation is then made from it on a glass slide, stained by a suitable method and then examined microscopically. The number of bacteria contained within a number of, say fifty, leucocytes can be counted and the average taken. In estimating the opsonic power of the serum in cases of disease a control with normal serum is made at the same time and under precisely the same conditions. The average number of bacteria contained within leucocytes in the case tested, divided by the number given by the normal serum, is called the phagocytic index. Wright and Douglas showed that under these conditions phagocytosis might occur when a small quantity of normal serum was present, whereas it was absent when normal salt solution was substituted for the serum; the latter thus contained substances which made the organisms susceptible to the action of the phagocytosis. They further showed that this substance acted by combining with the organisms and apparently producing some alteration in them; on the other hand it had no direct action on the leucocytes. This opsonin of normal serum is very labile, being rapidly destroyed at 55° C.; that is, a serum heated at this temperature has practically no greater effect in aiding phagocytosis than normal salt solution has. Various observers had previously found that the serum of an animal immunized against (a) Lysogenic action. a particular bacterium had a special action in bringing about phagocytosis of that organism, and it had been found that this property was retained when the serum was heated at 55° C. It is now generally admitted that at least two distinct classes of substances are concerned in opsonic action, that thermostable immune opsonins are developed as a result of active immunization and these possess the specific properties of anti-substances in general, that is, act only on the corresponding bacterium. On the contrary the labile opsonins of normal serum have a comparatively general action on different organisms. It is quite evident that the specific immune-opsonins may play a very important part in the phenomena of immunity, as by their means the organisms are taken up more actively by the phagocytic cells, and thereafter may undergo rapid disintegration.

The opsonic action of the serum has been employed by Sir A. Wright and his co-workers to control the treatment of bacterial infections by vaccines; that is, by injections of varying amounts of a dead culture of the corresponding bacterium. The object in such treatment is to raise the opsonic index of the serum, this being taken as an indication of increased immunity. The effect of the injection of a small quantity of vaccine is usually to produce an increase in the opsonic index within a few days. If then an additional quantity of vaccine be injected there occurs a fall in the opsonic index (negative phase) which, however, is followed later by a rise to a higher level than before. If the amounts of vaccine used and the times of the injection are suitably chosen, there may thus be produced by a series of steps a rise of the opsonic index to a high level. One of the chief objects in registering the opsonic power in such cases is to avoid the introduction of additional vaccine when the opsonic index is low, that is, during the negative phase, as if this were done a further diminution of the opsonic action might result. The principle in such treatment by means of vaccines is to stimulate the general production of anti-substances throughout the body, so that these may be carried to the sites of bacterial growth, and aid the destruction of the organisms by means of the cells of the tissues. A large number of favourable results obtained by such treatment controlled by the observation of the opsonic index have already been published, but it would be unwise at present to offer a decided opinion as to the ultimate value of the method.

Active immunity has thus been shown to be associated with the presence of certain anti-substances in the serum. After these substances have disappeared, however, as they always do in the course of time, the animal still possesses immunity for a varying period. This apparently depends upon some alteration in the cells of the body, but its exact nature is not known.

The destruction of bacteria by direct cellular agency both in natural and acquired immunity must not be overlooked.

The behaviour of certain cells, especially leucocytes, with regard to anti-bacterial sera, the presence of phagocytosis cannot be regarded as the essence of immunity, but rather the evidence of its existence. The increased ingestion of bacteria in active immunity would seem to depend upon the presence of immune opsonins in the serum. These, as already explained, are true anti-substances. Thus the apparent increased activity of the leucocytes is due to a preliminary effect of the opsonins on the bacteria. We have no distinct proof that there occurs in active immunity any education of the phagocytes, in Metchnikoff's sense, that is, any increase of the inherent ingestive or digestive activity of these cells. There is some evidence that in certain cases anti-substances may act upon the leucocytes, and to these the name of " stimulins " has been given. We cannot, however, say that these play an important part in immunity, and even if it were so, the essential factor would be the development of the substances which act in this way. While in immunity there probably occurs no marked change in the leucocytes themselves, it must be admitted that the increased destruction of bacteria by these cells is of the highest importance. This, as already pointed out, depends upon the increase of opsonins, though it is also to be noted that in many infective conditions there is another factor present, namely a leucocytosis, that is, an increase of the leucocytes in the blood, and the defensive powers of the body are thereby increased. Evidence has been brought forward within recent years that the leucocytes may constitute an important source of the antagonistic substances which appear in the serum. Much of such evidence possesses considerable weight, and seeing that these cells possess active digestive powers it is by no means improbable that substances with corresponding properties may be set free by them. To ascribe such powers to them exclusively is, however, not justifiable. Probably the lining endothelium of the blood-vessels as well as other tissues of the body participate in the production of anti-substances.

The subject of artificial immunity has occupied a large proportion of bacteriological literature within recent years, and our endeavour has been mainly to indicate the general laws which are inrocess of evolution. When the process facts of natural immunity are examined, we find that no single explanation is possible. Natural immunity against toxins must be taken into account, and, if Ehrlich's view with regard to toxic action be correct, this may depend upon either the absence of chemical affinity of the living molecules of the tissues for the toxic molecule, or upon insensitiveness to the action of the toxophorous group. It has been shown with regard to the former, for example, that the nervous system of the fowl, which possesses immunity against tetanus toxin, has little combining affinity for it. The non-sensitiveness of a cell to a toxic body when brought into immediate relationship cannot, however, be explained further than by saying that the disintegrative changes which underlie symptoms of poisoning are not brought about. Then as regards natural powers of destroying bacteria, phagocytosis aided by chemiotaxis plays a part, and it can be understood that an animal whose phagocytes are attracted by a particular bacterium will have an advantage over one in which this action is absent. Variations in chemiotaxis towards different organisms probably depend in natural conditions, as well as in active immunity, upon the opsonic content of the serum. Whether bacteria will be destroyed or not after they have been ingested by the leucocytes will depend upon the digestive powers of the latter, and these probably vary in different species of animals. The blood serum has a direct bactericidal action on certain bacteria, as tested outside the body, and this also varies in different animals. Observations made on this property with respect to the anthrax bacillus at first gave the hope that it might explain variations in natural immunity. Thus the serum of the white rat, which is immune to anthrax, kills the bacillus; whereas the serum of the guinea-pig, which is susceptible, has no such effect. Further observations, however, showed that this does not hold as a general law. The serum of the susceptible rabbit, for example, is bactericidal to this organism, whilst the serum of the immune dog is not. In the case of the latter animal the serum infective conditions led Metchnikoff to place great p g importance on phagocytosis. In this process there are two factors concerned, viz. the ingestion of bacteria by the cells, and the subsequent intracellular digestion. If either of these is wanting or interfered with, phagocytosis will necessarily fail as a means of defence. As regards the former, leucocytes are guided chiefly by chemiotaxis, i.e. by sensitiveness to chemical substances in their surroundings - a property which is not peculiar to them but is possessed by various unicellular organisms, including motile bacteria. When the cell moves from a less to a greater degree of concentration, i.e. towards the focus of production, the chemiotaxis is termed positive; when the converse obtains, negative. This apparently purposive movement has been pointed out by M. Verworn to depend upon stimulation to contraction or the reverse. Metchnikoff showed that in animals immune to a given organism phagocytosis is present, whereas in susceptible animals it is deficient or absent. He also showed that the development of artificial immunity is attended by the appearance of phagocytosis; also, when an anti-serum is injected into an animal, the phagocytes which formerly were indifferent might move towards and destroy the bacteria. In the light of all the facts, however, especially those contains an opsonin which leads to phagocytosis of the bacillus, and the latter is then destroyed by the leucocytes. It is quite evident that bactericidal action as tested in vitro outside the body does not correspond to the degree of immunity possessed by the animal under natural conditions. We may say, however, that there are several factors concerned in natural immunity, of which the most important may be said to be the three following, viz. variations in the bactericidal action of the serum in vivo, variations in the chemiotactic or opsonic properties of the serum in vivo, and variations in the digestive properties of the leucocytes of the particular animal. It is thus evident that the explanation of natural immunity in any given instance may be a matter of difficulty and much complexity.

Authorities. - Bacteriological literature has become so extensive that it is impossible to give here references to original articles, even the more important. A number of these, giving an account of classical researches, were translated from French and German, and published by the New Sydenham Society under the title Microparasites in Disease: Selected Essays, in 1886. The following list contains some of the more important books published within recent years. Abbott, Principles of Bacteriology (7th ed., London, 1905); Crookshank, Bacteriology and Infective Diseases (with bibliography, 4th ed., London, 1896); Duclaux, Traite de microbiologie (Paris, 1899-1900); Eyre, Bacteriological Technique (Philadelphia and London, 1902); Flugge, Die Mikroorganismen (3rd ed., Leipzig, 1896); Fischer, Vorlesungen fiber Bakterien (2nd ed., Jena, 1902); Gunther, Einfiihrung in das Studium der Bakteriologie (6th ed., Leipzig, 1906); Hewlett, Manual of Bacteriology (2nd ed., London, 1902); Hueppe, Principles of Bacteriology (translation, London, 1899); Klein, Micro-organisms and Disease (3rd ed., London, 1896); Kolle and Wassermann, Handbuch der pathogenen Mikroorganismen (Jena, 1904) (supplements are still being published; this is the most important work on the subject); Lofler, Vorlesungen fiber die geschichtliche Entwickelung der Lehre von der Bacterien (Leipzig, 1887); M`Farland, Text-book upon the Pathogenic Bacteria (5th ed., London, 1906); Muir and Ritchie, Manual of Bacteriology (with bibliography, 4th ed., Edin. and Lond., 1908); Park, Pathogenic Micro-organisms (London, 5906); Sternberg, Manual of Bacteriology (with full bibliography, 2nd ed., New York, 1896); Woodhead, Bacteria and their products (with bibliography, London, 1891). The bacteriology of the infective diseases (with bibliography) is fully given in the System of Medicine, edited by Clifford Allbutt, (2nd ed., London, 1907). For references consult Centralbl. fiir Bakter. u. Parasitenk. (Jena); also Index Medicus. The most important works on immunity are: Ehrlich, Studies in Immunity (English translation, New York, 5906), and Metchnikoff, Immunity in Infective Diseases (English translation, Cambridge, 1905). (R. M.*)