The full description of a particular bacterium implies an account not only of its microscopical characters, but also of its growth characters in various culture media, its biological properties, and the effects produced in animals by inoculation. To demonstrate readily its action on various substances, certain media have been devised. For example, various sugars - lactose, glucose, saccharose, etc. - are added to test the fermentative action of the bacterium on these substances; litmus is added to show changes in reaction, specially standardized media being used for estimating such changes; peptone solution is commonly employed for testing whether or not the bacterium forms indol; sterilized milk is used as a culture medium to determine whether or not it is curdled by the growth. Sometimes a bacterium can be readily recognized from one or two characters, but not infrequently a whole series of tests must be made before the species is determined. As our knowledge has advanced it has become abundantly evident that the so-called pathogenic bacteria are not organisms with special features, but that each is a member of a group of organisms possessing closely allied characters.
From the point of view of evolution we may suppose that certain races of a group of bacteria have gradually acquired the power of invading the tissues of the body and producing disease. In the acquisition of pathogenic properties some of their original characters have become changed, but in many instances this has taken place only to a slight degree, and, furthermore, some of these changes are not of a permanent character. It is to be noted that in the case of bacteria we can only judge of organisms being of different species by the stability of the characters which distinguish them, and numerous examples might be given where their characters become modified by comparatively slight change in their environment. The cultural as well as the microscopical 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, etc., 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 Inoculation. 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 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, etc., 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.