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 uber Bakterien_.)]
[Illustration: FIG. 16.
a, root nodule of the lupin, nat. size. (From Woromv.)
b, longitudinal section through root and nodule.
g, fibro-vascular bundle.
w, bacterial tissue. (After Woromv.)
c, cell from bacterial tissues showing nucleus and protoplasm filled with bacteria.
d, bacteria from nodule of lupin, normal undegenerate form.
e and f, bacteroids from _Vicia villosa_ and _Lupinus albus_. (After Morck.)
(From Fischer's _Vorlesungen uber 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.
[Illustration: 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.)]
[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.
[Illustration: 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.
[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 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., 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.
[Illustration: FIG. 19.--Ginger-beer plant, showing yeast (_Saccharomyces pyriformis_) entangled in the meshes of the bacterium (_B. vermiforme_).
(H. M. W.)]
[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 100 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 (-192 C. to -183 C.) and again grow when put into normal conditions. More recent experiments have shown that even ten hours' exposure to the temperature of liquid hydrogen -252 C. (21 on the absolute scale) failed to kill them. It is probable that all these cases of resistance of seeds, spores, &c., are to be connected with the fact that completely dry albumin does not lose its coagulability on heating to 110 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.]