Chapter II: Front Matter (2)

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 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 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. Muentz 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 [v.03 p.0165] 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 OH_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, 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.

[Sidenote: Bacteria and Leguminosae.]

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.

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's _Vorlesungen ueber 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.

e and f, bacteroids from _Vicia villosa_ and _Lupinus albus_. (After Morck.)

(From Fischer's _Vorlesungen ueber 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 Maze that the bacterium can even absorb free nitrogen when grown in cultures [v.03 p.0166] 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 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.

[Sidenote: Cellulose-bacteria.]

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.

[Sidenote: Sulphur bacteria.]

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 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 [v.03 p.0167] 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.

[Sidenote: Iron bacteria.]

Chalybeate waters, pools in marshes near ironstone, &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 + 3OH_2 + O = Fe_2(OH)_6 + 2CO_2.

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.

[Sidenote: Pigment bacteria.]

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 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 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 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 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.

[Sidenote: Dairy bacteria.]

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, 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.

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.

[v.03 p.0168]

[Sidenote: Thermophilous bacteria.]

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, 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 70deg 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 70deg C., and _B. Ludwigi_ and _B. ilidzensis_, &c., from hot springs, &c.

[Sidenote: Phosphorescent bacteria.]

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 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.

[Sidenote: Oxidizing bacteria.]

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 OH_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. kutzingianum_, 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 OH_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.

[Sidenote: Bacteria and light.]

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 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 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. anthracis_, which withstand temperatures of 100deg 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 light-action; 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 [v.03 p.0169] 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.

[Sidenote: Bacteria and cold.]

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 (-192deg C. to -183deg 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 -252deg C. (21deg 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 110deg C. for some hours, since it is well known that completely ripe spores and dry heat are the conditions of extreme experiments.

[Sidenote: Pathogenic bacteria.]

No sharp line can be drawn between pathogenic and non-pathogenic Schizomycetes, and some of the most marked steps in the progress of our modern knowledge of these organisms depend on the discovery that their pathogenicity 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.

A. One of the brain-like gelatinous masses into which the mature "plant" 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., c at 9 P.M., d at 11 A.M. next day, e at 3 P.M., f at 9 P.M., g at 10.30 A.M. next day, h at 24 hours later. (H. M. W.)]

[Sidenote: Bacteriosis in plants.]

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 cause of the disease referred to their agency. Until 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 Beyerinck's discovery that the juices of tobacco plants affected with the disease known as "leaf mosaic," will induce this disease after filtration through porcelain.

[Sidenote: Symbiosis.]

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 arid, inactive lactic acid, carbon dioxide, and 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 [v.03 p.0170] 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_.

[Sidenote: Activity of bacteria.]

Astonishment has been frequently expressed at the powerful activities of bacteria--their rapid growth and dissemination, the extensive and profound decompositions and 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 in more complex multi-cellular organisms. To put the matter in another way, if we could imagine all the living cells of a large oak or of a horse, having given up the specializations of function 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 9-10 cm. or more in 15 minutes--say 0.1 mm. or 100 [micron] per second--appears to be darting across the field with great velocity, because its own small size--say 5 x 1 [micron]--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. It is usually stated that the carbon dioxide molecule is here 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._, 1893, p. 154) 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 natuerlichen Pflanzenfamilien_, I. Th. 1 Abt. a; Lafar, _Technical Mycology_ (vol. i. London, 1898); Mace, _Traite pratique de bakteriologie_ (5th ed. 1904). Fossil bacteria: Renault, "Recherches sur les Bacteriacees 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. 1; 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.: Butschli, _Weitere Ausfuhrungen ueber den Bau der Cyanophyceen und Bakterien_ (Leipzig, 1896); Fischer, _Unters. ueber den Bau der Cyanophyceen und Bakterien_ (Jena, 1897); Rowland, "Observations upon the Structure of Bacteria," _Trans. Jenner Institute_, 2nd ser. 1899, p. 143, with literature. Cilia: Fischer, "Unters. ueber 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, "Ueber den Kern der Bakterien und seine Teilung," _Cent. f. Bakt._ Abt. II. Bd. xi. (1904) p. 481; _ibid._ "Cytologisches ueber die Bakterien der Prager Wasserleitung," _Cent. f. Bakt._ Abt. II. Bd. xv. (1905); Mencl, "Nachtraege zu den Strukturverhaeltnissen 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. 1; Sturgis, "A Soil Bacillus of the type of de Bary's _B. megatherium_," _Phil. Trans._ [v.03 p.0171] vol. cxci. p. 147; Klein, L., _Ber. d. deutschen bot. Gesellsch._ (1889), Bd. vii.; and _Cent. f. Bakt. und 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, and "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. lviii. 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. 1 (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, "Unters. ueber die Stickstoffnahrung der Gramineen u. Leguminosen," _Beit. Zeit. d. Vereins fuer die Ruebenzuckerindustrie_ (Berlin, 1888); Nobbe and Hiltner, _Landw. Versuchsstationen_ (1899), Bd. 51, p. 241, and Bd. 52, p. 455; Maze, _Annales de l'Institut Pasteur_, t. II, 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. 441; Omelianski, "Sur la fermentation de la cellulose," _Compt. Rend._, 4 Nov. 1895; van Senus, _Beitr. zur Kenntn. der Cellulosegaehrung_ (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 Evolution 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._, 1893, 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 Immunitaets- und Spezifizetaetslehre 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.)

II. 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.

[Sidenote: Historical summary.]

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 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 "plate-method," &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 Gruenbaum 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 sense--was only of service in producing immunity as a preventive measure against the corresponding organism, but the work of [v.03 p.0172] 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.