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172
BACTERIOLOGY

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.

The methods employed in studying the relation of bacteria to disease are in principle comparatively simple, but considerable experience and great care are necessary in applying them and in interpreting results. In any given Methods
of study.
disease there are three chief steps, viz. (1) the discovery of a bacterium in the affected tissues by means of the microscope; (2) the obtaining of the bacterium in pure culture; and (3) the production of the disease by inoculation with a pure culture. 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 methods vary much in detail, though in each case the endeavour is to colour the bacteria as deeply, and the tissues as faintly, as possible. Sometimes a simple watery solution of the dye is sufficient, but very often the best result is obtained by increasing the staining power, e.g. by addition of weak alkali, application of heat, &c., and by using some substance which acts as a mordant and tends to fix the stain to the bacteria. Excess of stain is afterwards removed from the tissues by the use of decolorizing agents, such as acids of varying strength and concentration, alcohol, &c. Different bacteria behave very differently to stains; some take them up rapidly, others slowly, some resist decolorization, others are easily decolorized. In some instances the stain can be entirely removed from the tissues, leaving the bacteria alone coloured, and the tissues can then be stained by another colour. This is the case in the methods for staining the tubercle bacillus and also in Gram’s method, the essential point in which latter is the treatment with a solution of iodine before decolorizing. In Gram’s method, however, only some bacteria retain the stain, while others lose it. The tissues and fluids are treated by various histological methods, but, to speak generally, examination is made either in films smeared on thin cover-glasses and allowed to dry, or in thin sections cut by the microtome after suitable fixation and hardening of the tissue. In the case of any bacterium discovered, observation must be made in a long series of instances in order to determine its invariable presence.

In cultivating bacteria outside the body various media to serve as food material must be prepared and sterilized by heat. The general principle in their preparation is to supply the nutriment for bacterial growth in Cultivation.a form as nearly similar as possible to that of the natural habitat of the organisms—in the case of pathogenic bacteria, the natural fluids of the body. The media are used either in a fluid or solid condition, the latter being obtained by a process of coagulation, or by the addition of a gelatinizing agent, and are placed in glass tubes or flasks plugged with cotton-wool. To mention examples, blood serum solidified at a suitable temperature is a highly suitable medium, and various media are made with extract of meat as a basis, with the addition of gelatine or agar as solidifying agents and of non-coagulable proteids (commercial “peptone”) to make up for proteids lost by coagulation in the preparation. The reaction of the media must in every case be carefully attended to, a neutral or slightly alkaline reaction being, as a rule, most suitable; for delicate work it may be necessary to standardize the reaction by titration methods. The media from the store-flasks are placed in glass test-tubes or small flasks, protected from contamination by cotton-wool plugs, and are sterilized by heat. For most purposes the solid media are to be preferred, since bacterial growth appears as a discrete mass and accidental contamination can be readily recognized. Cultures are made by transferring by means of a sterile platinum wire a little of the material containing the bacteria to the medium. The tubes, after being thus inoculated, are kept at suitable temperatures, usually either at 37° C., the temperature of the body, or at about 20° C., a warm summer temperature, until growth appears. For maintaining a constant temperature incubators with regulating apparatus are used. Subsequent cultures or, as they are called, “subcultures,” may be made by inoculating fresh tubes, and in this way growth may be maintained often for an indefinite period. The simplest case is that in which only one variety of bacterium is present, and a “pure culture” may then be obtained at once. When, however, several species are present together, means must be adopted for separating them. For this purpose various methods have been devised, the most important being the plate-method of Koch. 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. Of course, in applying the method means must be adopted for suitably diluting the bacterial mixture. Another important method consists in inoculating an animal with some fluid containing the various bacteria. A pathogenic bacterium present may invade the body, and may be obtained in pure culture from the internal organs. This method applies especially to pathogenic bacteria whose growth on culture media is slow, e.g. the tubercle bacillus.

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, &c.—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