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 Methods of study. 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 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, etc., 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, etc. 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 Cultivation. 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 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.