Harry Marshall Ward, Vernon Herbert Blackman and Robert Muir

“ The phenomenon of agglutination depends essentially on the union of molecules in the bacteria—the agglutinogens—with the corresponding agglutinins, but another essential is the presence of a certain amount of salts in the fluid, as it can be shown that when agglutinated masses of bacteria are washed salt-free the clumps become resolved. The fact that agglutinins appear in the body at an early stage in a disease has been taken by some observers as indicating that they have nothing to do with immunity, their development being spoken of as a reaction of infection. ”

“ In still another class of diseases the bacteria are restricted to some particular part of the body, and the symptoms are due to toxins which are absorbed from it. Thus in cholera the bacteria are practically confined to the intestine, in diphtheria to the region of the false membrane, in tetanus to some wound. In the last-mentioned disease even the local multiplication depends upon the presence of other bacteria, as the tetanus bacillus has practically no power of multiplying in the healthy tissues when introduced alone. ”

“ According to this view, then, a part at least of the directly toxic substance is produced in the living body by enzymes present in the so-called toxin obtained from the bacterial culture. Recent researches go to show that enzymes play a greater part in fermentation by living ferments than was formerly supposed, and by analogy it is likely that they are also concerned in the processes of disease. But this has not been proved, and hitherto no enzyme has been separated from a pathogenic bacterium capable of forming, by digestive or other action, the toxic bodies from proteids outside the body. ”

“ 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. ”

“ The non-sensitiveness of a cell to a toxic body when brought into immediate relationship cannot, however, be explained further than by saying that the disintegrative changes which underlie symptoms of poisoning are not brought about. Then as regards natural powers of destroying bacteria, phagocytosis aided by chemiotaxis plays a part, and it can be understood that an animal whose phagocytes are attracted by a particular bacterium will have an advantage over one in which this action is absent. ”

“ 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. ”

“ The increased ingestion of bacteria in active immunity would seem to depend upon the presence of immune opsonins in the serum. These, as already explained, are true anti-substances. Thus the apparent increased activity of the leucocytes is due to a preliminary effect of the opsonins on the bacteria. We have no distinct proof that there occurs in active immunity any education of the phagocytes, in Metchnikoff’s sense, that is, any increase of the inherent ingestive or digestive activity of these cells. ”

“ The virulence of many organisms, however, becomes diminished when they are grown on the ordinary artificial media, and the diminution is sometimes accelerated by passing a current of air over the surface of the growth. Sometimes also the virulence of a bacterium for a particular kind of animal becomes lessened on passing it through the body of one of another species. Cultures of varying degree of virulence may be obtained by such methods, and immunity can be gradually increased by inoculation with vaccines of increasing virulence. ”

“ By rapid division hundreds of thousands of cells may be produced in a few hours, [3] and, according to the species and the conditions (the medium, temperature, &c.) , enormous collections of isolated cells may cloud the fluid in which they are cultivated, or form deposits below or films on its surface; valuable characters are sometimes obtained from these appearances. When these dense “swarms” of vegetative cells become fixed in a matrix of their own swollen contiguous cell-walls, they pass over into a sort of resting state as a so-called zoogloea (fig. 3) . ”

“ 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. ”

“ In this way conditions formerly believed to be the causes of disease are now recognized as playing their part in predisposing to the action of the true causal agent, viz. the bacterium. In health the blood and internal tissues are bacterium-free; after death they offer a most suitable pabulum for various bacteria; but between these two extremes lie states of varying liability to infection. It is also probable that in a state of health organisms do gain entrance to the blood from time to time and are rapidly killed off. ”

“ 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. ”

“ By immunity is meant non-susceptibility to a given disease, or to experimental inoculation with a given bacterium or toxin. The term must be used in a relative sense, and account must always be taken of the conditions present. Immunity.An animal may be readily susceptible to a disease on experimental inoculation, and yet rarely or never suffer from it naturally, because the necessary conditions of infection are not supplied in nature. That an animal possesses natural immunity can only be shown on exposing it to such conditions, this being usually most satisfactorily done in direct experiment. ”

“ In certain instances, e.g. in the case of the tetanus and diphtheria bacilli, the production of soluble toxins can be readily demonstrated by filtering a culture in bouillon germ-free by means of a porcelain filter, and then injecting some of the filtrate into an animal. In this way the characteristic features of the disease can be reproduced. Such toxins being set free in the culture medium are often known as extracellular. In many cases, however, the filtrate, when injected, produces comparatively little effect, whilst toxic action is observed when the bacteria in a dead condition are used ”

“ In this method the bacteria are distributed in a gelatine or agar medium liquefied by heat, and the medium is then poured out on sterile glass plates or in shallow glass dishes, and allowed to solidify. Each bacterium capable of growth gives rise to a colony visible to the naked eye, and if the colonies are sufficiently apart, an inoculation can be made from any one to a tube of culture-medium and a pure culture obtained. ”

“ 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. ”

“ Susceptibility, as is well recognized, varies much under natural conditions in different species, in different races of the same species, and amongst individuals of the same race. It also varies with the period of life, young subjects being more susceptible to certain diseases, e.g. diphtheria, than adults. Further, there is the very important factor of acquired susceptibility. It has been experimentally shown that conditions such as fatigue, starvation, exposure to cold, &c., lower the general resisting powers and increase the susceptibility to bacterial infection. ”

“ By means of microscopic examination more than one organism may sometimes be observed in the tissues, but one single organism by its constant presence and special relations to the tissue changes can usually be selected as the probable cause of the disease, and attempts towards its cultivation can then be made. Such microscopic examination requires the use of the finest lenses and the application of various staining methods. In these latter the basic aniline dyes in solution are almost exclusively used, on account of their special affinity for the bacterial protoplasm. ”

“ The 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, however, also accompanied by the appearance of certain bodies in the blood serum of the animal treated, to which the name of anti-substances is given, and these have been the subject of extensive study. It is by means of them that immunity (passive) can be transferred to a fresh animal. The development of anti-substances is, however, not peculiar to bacteria, but occurs also when alien cells of various kinds, proteins, ferments, &c., are injected. In fact, organic molecules can be divided into two classes according as they give rise to anti-substances or fail to do so. ”

“ Of the chief methods used in producing active immunity the first is by inoculation with bacteria whose virulence has been diminished, i.e. with an “attenuated virus.” Many of the earlier methods of attenuation were devised in the case of the anthrax bacillus, an organism which is, however, somewhat exceptional as regards the relative stability of its virulence. Many such methods consist, to speak generally, in growing the organism outside the body under somewhat unsuitable conditions, e.g. at higher temperatures than the optimum, in the presence of weak antiseptics, &c. ”

“ Metchnikoff showed that in animals immune to a given organism phagocytosis is present, whereas in susceptible animals it is deficient or absent. He also showed that the development of artificial immunity is attended by the appearance of phagocytosis; also, when an anti-serum is injected into an animal, the phagocytes which formerly were indifferent might move towards and destroy the bacteria. ”

“ 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. ”

“ It may be stated that the introduction of a particular bacterium into the tissues of the body leads to certain properties appearing in the serum, which are chiefly exerted towards this particular bacterium. Such a serum may accordingly within certain limits be used for differentiating this organism from others closely allied to it (vide infra) .
The modes of cultivation described apply only to organisms which grow in presence of oxygen. Some, however—the strictly anaerobic bacteria—grow only in the absence of oxygen ”

“ 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. ”

“ The serum of the susceptible rabbit, for example, is bactericidal to this organism, whilst the serum of the immune dog is not. In the case of the latter animal the serum contains an opsonin which leads to phagocytosis of the bacillus, and the latter is then destroyed by the leucocytes. It is quite evident that bactericidal action as tested in vitro outside the body does not correspond to the degree of immunity possessed by the animal under natural conditions. ”

“ It appears from the observations of Mazé that the bacterium can even absorb free nitrogen when grown in cultures 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. ”

“ 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. ”

“ This conclusion is not justified, as we must suppose that the process of immunization begins to be developed at an early period in the disease, that it gradually increases, and ultimately results in cure. It should also be stated that agglutinins are used up in the process of agglutination, apparently combining with some element of the bacterial structure. In view of all the facts it must be admitted that the agglutinins and immune bodies are the result of corresponding reactive processes, and are probably related to one another. ”