Chapter 2

Fig. 9. Filaments of Bacillus spp.Fig.9.A.Bacillus anthracis.(After de Bary) Two of the long filaments (B, fig. 10) 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.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.

A.Bacillus anthracis.(After de Bary) Two of the long filaments (B, fig. 10) 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.

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.

Fig. 10. Bacillus subtilis.Fig.10.—Bacillus subtilis. (After Strasburger.) A. Zoogloea pellicle. B. Motile rodlets. C. Development of spores.

Fig.10.—Bacillus subtilis. (After Strasburger.) A. Zoogloea pellicle. B. Motile rodlets. C. Development of spores.

The spore may be formed in short or long segments, the cell-wall 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 "Köpfchenbacterien" of older writers are simply bacterioid segments with a spore at one end, the mother cell-wall having adapted itself to the outline of the spore (fig. 4, F). The ripe spores of Schizomycetes are spherical, ovoid or long-ovoid in shape and extremely minute (e.g.those ofBacillus subtilismeasure 0.0012 mm. long by 0.0006 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. 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 11); 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 so-called 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.

Fig. 11. Development of spores of Bacillus ramosus.Fig.11.—Stages in the development of spores ofBacillus 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.)

Fig.11.—Stages in the development of spores ofBacillus 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.)

The difficult subject of the classification[4]Classification.of bacteria dates 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 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 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 nutritionand 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 Miquel (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.Fischer's Scheme.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. 1) 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.Order—Haplobacterinae.Vegetative body unicellular; spheroidal, cylindrical or spirally twisted; isolated or connected in filamentous or other growth series.

1.Family—Coccaceae. 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.

(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).(b) Sub-family—Clostridieae. Sporogenous rodlets, spindle-shaped:—Clostridium(Prazm.), motile (peritrichous).(c) Sub-family—Plectridieae. Sporogenous rodlets, drumstick-shaped:—Plectridium(Fischer), motile (peritrichous).

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

3.Family—Spirillaceae. Vegetative cells, cylindric but curved more or less spirally. Divisions perpendicular to the long axis:—Vibrio(Müller-Löffler), comma-shaped, motile, monotrichous;Spirillum(Ehrenb.), more strongly curved in open spirals, motile, lophotrichous;Spirochaete(Ehrenb.), spirally coiled in numerous close turns, motile, but apparently owing to flexile movements, as no cilia are found.

II.Order—Trichobacterinae.Vegetative body of branched or unbranched cell-filaments, the segments of which separate as swarm-cells (Gonidia).

1.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-dichotomous manner.(b) Filaments showing slow pendulous and creeping movements, and with no distinct sheath:—Beggiatoa(Trev.), with sulphur particles.

(b) Filaments showing slow pendulous and creeping movements, and with no distinct sheath:—Beggiatoa(Trev.), 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. megatheriumandB. subtilispass 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 1000) fall into the three genera—Micrococcus(about 400),Bacillus(about 200) andBactridium(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; thusPseudomonas rosea(Mig.) may have 1, 2 or 3 cilia at either end, and would be distributed by Fischer's classification betweenBactriniumandBactrillum, 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.

Fig. 12. Myxobacteriaceae.Fig.12.A.Myxococcus digelatus, bright red fructification occurring on dung.B.Polyangium primigenum, red fructification on dog's dung.C.Chondromyces apiculatus, orange fructification on antelope's dung.D. Young fructification.E. Single cyst germinating.(A, B, after Quehl; C-E, after Thaxter.) From Strasburger'sLehrbuch der Botanik, by permission of Gustav Fischer.

A.Myxococcus digelatus, bright red fructification occurring on dung.

B.Polyangium primigenum, red fructification on dog's dung.

C.Chondromyces apiculatus, orange fructification on antelope's dung.

D. Young fructification.

E. Single cyst germinating.

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

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 asMyxobacter, was shown later to be the same asPolyangium vitellinumdescribed 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 plasmodium-like 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 butinimical to that of the fungi which are so common on this substratum.

The discoveries that some species of nitrifying bacteria andFunction and life of bacteria.perhaps pigmented forms are capable of carbon-assimilation, that others can fix free nitrogen and that a number of decompositions hitherto unsuspected are accomplished by Schizomycetes, have put the questions of 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 many are indispensable aids in natural operations important to man.

Fig. 13. Germination of the spore of Bacillus ramosus.Fig.13.—A series of phases of germination of the spore ofB. ramosussown 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.)

Fig.13.—A series of phases of germination of the spore ofB. ramosussown 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.)

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, sulphur- and 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 themPrototrophic. 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 importantrôlein the evolution of life on our globe. The vast majority of bacteria, on the other hand, which are ordinarily termed saprophytes, aresaprogenic,i.e.bring organic material to the putrefactive state—orsaprophilous,i.e.live best in such putrefying materials—or becomezymogenic,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 FischerMetatrophic, 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,Paratrophicbacteria, 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 ofNitrogen bacteria.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 Miquel 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 débris. 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 thatClostridium 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. Müntz 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 theNitroso-bacteria, is only capable of bringing about the oxidation of the ammonia to nitrous acid, and the astonishing result was obtained thatthis 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 CO2and OH2. TheNitro-bacteriaare 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, 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.

Fig. 14. Formation of a colony of Bacillus vulgaris.Fig.14.—Stages in the formation of a colony of a variety ofBacillus (Proteus) vulgaris(Hauser), observed in a hanging drop. At 11A.M.a rodlet appeared (A); at 4P.M.it had grown and divided and broken up into eight rodlets (B); C shows further development at 8P.M., D at 9.30P.M.—all under a high power. At E, F, and G further stages are drawn, as seen under much lower power. (H. M. W.)

Fig.14.—Stages in the formation of a colony of a variety ofBacillus (Proteus) vulgaris(Hauser), observed in a hanging drop. At 11A.M.a rodlet appeared (A); at 4P.M.it had grown and divided and broken up into eight rodlets (B); C shows further development at 8P.M., D at 9.30P.M.—all under a high power. At E, F, and G further stages are drawn, as seen under much lower power. (H. M. W.)

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,Bacteria and Leguminosae.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 anormalcharacteristic 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.

Fig. 15. Invasion of leguminous roots by bacteria.Fig.15.—Invasion of leguminous roots by bacteria.a, cell from the epidermis of root of Pea with "infection thread" (zoogloea) pushing its way through the cell-walls. (After Prazmowski.)b, 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'sVorlesungen über Bakterien.)

Fig.15.—Invasion of leguminous roots by bacteria.

a, cell from the epidermis of root of Pea with "infection thread" (zoogloea) pushing its way through the cell-walls. (After Prazmowski.)

b, 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'sVorlesungen über Bakterien.)

Fig. 16. Bacteria in legume roots.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.eandf, bacteroids fromVicia villosaandLupinus albus. (After Morck.)(From Fischer'sVorlesungen über Bakterien.)

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.

eandf, bacteroids fromVicia villosaandLupinus albus. (After Morck.)

(From Fischer'sVorlesungen über Bakterien.)

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 Mazé that the bacterium can even absorb free nitrogen when grown in culturesoutside 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 so-called "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.

The apparent specialization 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 outside this particular group. However, Professor Bottomley announced 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.

Fig. 17. Effect of light on bacillus.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.)

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.)

Another important advance is in our knowledge of the partCellulose-bacteria.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 observedSulphur bacteria.in the cells ofBeggiatoaconsist of sulphur, and Winogradsky and Beyerinck have shown that a whole series of sulphur bacteria of the generaThiothrix,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 marsh gas, the nascent gas reduces sulphates—e.g.gypsum—with liberation of SH2, and it is found that the sulphur bacteria thrive under such conditions by oxidizing the SH2and storing the sulphur in their own protoplasm. If the SH2runs 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 SH2is 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 SH2. In the deeper parts of this zone the bacteria absorb the SH2, and, as they rise, oxidize it and store up the sulphur; then ascending into planes more highly oxygenated, oxidize the sulphur to SO3. These bacteria therefore employ SH2as 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 thatSpirillum 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, takeninto the higher plants as sulphates, built up into proteids, decomposed by putrefactive bacteria and yielding SH2which the sulphur bacteria oxidize, the resulting sulphur is then again oxidized to SO3and again combined with calcium to gypsum, the cycle being thus complete.

Chalybeate waters, pools in marshes near ironstone, &c,Iron bacteria.abound in bacteria, some of which belong to the remarkable generaCrenothrix,CladothrixandLeptothrix, 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—

2FeCO3+ 3OH2+ O = Fe2(OH)6+ 2CO2.

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 chromogenicPigment bacteria.bacteria have been made by the studies of Beyerinck, Lankester, Engelmann, Ewart and others, and have assumed exceptional importance owing to the discovery thatBacteriopurpurin—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 bacteriopurpurin 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 byB. pyocyaneus,B. fluorescensand a whole group of fluorescent bacteria. Neelson found that the pigment ofB. cyanogenusgives 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 ofB. prodigiosus,B. ruber, &c., the violet ofB. violacens,B janthinus, &c., the red-purple 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 yellowMicrococcus cereus flavusand theB. 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 ofDairy bacteria.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, 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. Effect of light spectra on bacillus.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.)

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.

That bacterial fermentations are accompanied by the evolutionThermophilous bacteria.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, 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 Miquel, 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° C., andB. LudwigiandB. ilidzensis, &c., from hot springs, &c.

The bodies of sea fish,e.g.mackerel and other animals, havePhosphorescent bacteria.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 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 termedPhotobacterium phosphorescensby Beyerinck will absorb maltose, and will become luminous if that sugar is present, whereasP. Pflugeriis 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 containingP. phosphorescenswill become luminous; if the latter, only those containingP. 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 isMicrococcus 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 dependsOxidizing bacteria.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 CO2and OH2; 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. kutzingianum, andB. 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 CO2and OH2supports 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.

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