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173
BACTERIOLOGY

characters of a pathogenic organism may be closely similar to other non-pathogenic members of the same group, and it thus comes to be a matter of extreme difficulty in certain cases to state what criterion should be used in differentiating varieties. The tests which are applied for this purpose at present are chiefly of two kinds. In the first place, such organisms may be differentiated by the chemical change produced by them in various culture media, e.g. by their fermentative action on various sugars, &c., though in this case such properties may become modified in the course of time. And in the second place, the various serum reactions to be described below have been called into requisition. It may be stated that the introduction of a particular bacterium into the tissues of the body leads to certain properties appearing in the serum, which are chiefly exerted towards this particular bacterium. Such a serum may accordingly within certain limits be used for differentiating this organism from others closely allied to it (vide infra).

The modes of cultivation described apply only to organisms which grow in presence of oxygen. Some, however—the strictly anaerobic bacteria—grow only in the absence of oxygen; hence means must be adopted for excluding this gas. It is found that if the inoculation be made deep down in a solid medium, growth of an anaerobic organism will take place, especially if the medium contains some reducing agent such as glucose. Such cultures are called “deep cultures.” To obtain growth of an anaerobic organism on the surface of a medium, in using the plate method, and also for cultures in fluids, the air is displaced by an indifferent gas, usually hydrogen.

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

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

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