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B A C T E R I O L 0 G Y 63 increasing the staining power, e.y., by addition of weak Another important method consists in inoculating an animal alkali, application of heat, ifcc., and by using some sub- with some fluid containing the various bacteria. A pathostance which acts as a mordant and tends to fix the stain genic bacterium present may invade the body, and may be to the bacteria. Excess of stain is afterwards removed obtained in pure culture from the internal organs. This from the tissues by the use of decolourizing agents, such method applies especially to pathogenic bacteria whose as acids of varying strength and concentration, alcohol, growth on culture media is slow, e.g., the tubercle bacillus. &c. Different bacteria behave very differently to stains; The full description of a particular bacterium implies some take them up rapidly, others slowly, some resist an account not only of its microscopical characters, but decolourization, others are easily decolourized. In some also of its growth characters in various culture media, its instances the stain can be entirely removed from the biological properties, and the effects produced in animals tissues, leaving the bacteria alone coloured, and the tissues by inoculation. To demonstrate readily its action on can then be stained by another colour. This is the case various substances, certain media have been devised. in the methods for staining the tubercle bacillus and also For example, lactose and the other sugars are added to in Gram’s method, the essential point in which latter is the test the action of the bacterium on these substances; treatment with a solution of iodine before decolourizing. In litmus is added to show changes in reaction, specially Gram’s method, however, only some bacteria retain the stain, standardized media being used for estimating such while others lose it. The tissues and fluids are treated by changes; peptone solution is commonly employed for various histological methods, but, to speak generally, ex- testing whether or not the bacterium forms indol; sterilamination is made either in films smeared on thin cover- ized milk is used as a culture medium to determine glasses and allowed to dry, or in thin sections cut by the whether or not it is curdled by the growth. Sometimes microtome after suitable hardening and fixation of the a bacterium can be readily recognized from one or two tissue. In the case of any bacterium discovered, observa- characters, but not infrequently a whole series of tests tion must be made in a long series of instances in order to must be made before the species is determined; this is, determine its invariable presence. for example, the case with members of the bacillus coli In cultivating bacteria outside the body various media group. to serve as food material must be prepared and sterilized The modes of cultivation described apply only to e at organisms which grow in presence of oxygen. Some, Cultivanh. - The general principle in their preparation. ti° is f° supply the nutriment in a form as however—the strictly anaerobic bacteria—grow only in nearly similar as possible to that of the natural the absence of oxygen, hence means must be adopted for habitat of the bacterium—in the case of pathogenic bacteria, excluding this gas. It is found that if the inoculation be the natural fluids of the body. The media are used either made deep down in a solid medium, growth of an anaerobic in a fluid or solid condition, the latter being obtained by organism will take place, especially if the medium contains a process of coagulation, or by the addition of a gelatiniz- some reducing agent such as glucose. Such cultures are ing agent, and are placed in glass tubes or flasks plugged called “ deep cultures.” To obtain growth of an anaerobic with cotton-wool. To mention examples, blood serum organism on the surface of a medium, in using the plate solidified at a suitable temperature is a highly suitable method, and also for cultures in fluids, the air is displaced medium, and various media are made with extract of meat by an indifferent gas, usually hydrogen. as. a basis, with the addition of gelatine or agar as solidiIn testing the effects of bacteria by inoculation the fyiflg agents and of non-coagulable proteids (commercial smaller rodents, rabbits, guinea - pigs, and mice, are “ peptone ”) to make up for proteids lost by coagulation in usually employed. One great drawback in the preparation. The reaction of the media must in every certain cases is that such animals are not ,aocul*m case be carefully attended to, a neutral or slightly alkaline susceptible to a given bacterium, or that the tl°a' reaction being, as a rule, most suitable. The media from disease is different in character from that in the human the store-flasks are placed in glass test-tubes or small flasks, subject. In some cases, e.g., Malta fever and relapsing protected from contamination by cotton-wool plugs, and fever, monkeys have been used with success, but in are sterilized by heat. As a rule the solid media are to be others, e.g., leprosy, none of the lower animals have been preferred, since bacterial growth appears as a discrete mass found to be susceptible. Discretion must therefore be and accidental contamination can be readily recognized. exercised in interpreting negative results in the lower Cultures are made by transferring by means of a sterile animals. For purposes of inoculation young vigorous platinum wire a little of the material containing the bacteria cultures must be used. The bacteria are mixed with to the medium. The tubes, after being thus inoculated, some indifferent fluid, or a fluid culture is employed. are kept at suitable temperatures, usually either at 37° C., The injections are made by means of a hypodermic syringe the temperature of the body, or at about 20° C., a warm into the subcutaneous tissue, into a vein, into one of the summer temperature. For maintaining a constant tempera- serous sacs, or more rarely into some special part of the ture incubators with regulating apparatus are used. The body. The animal, after injection, must be kept in simplest case is that in which only one variety of bacterium is present, and a “ pure culture ” may then be obtained at favourable surroundings, and any resulting symptoms noted. It may die, or may be killed at any time desired, once. When, however, several species are present together, and then a post-mortem examination is made, the conmeans must be adopted for separating them. For this of the organs, &c., being observed and noted. purpose various methods have been devised, the most im- ditions The various tissues affected are examined microscopically portant being the plate-method of Koch. In this method and cultures made from them; in this way the structural the bacteria are distributed in a gelatine or agar medium liquefied by heat, and the medium is then poured out on changes and the relation of bacteria to them can be sterile glass plates or in shallow glass dishes, and allowed determined. Though the causal relationship of a bacterium to a o solidify. Each bacterium capable of growth gives rise o a colony visible to the naked eye, and, if the colonies disease may be completely established by the methods are sufficiently apart, an inoculation can be made from given, another very important part of bacteriology is any one to a tube of culture-medium and a pure culture concerned with the poisons or toxins formed by bacteria. Ihese toxins may become free in the culture fluid, and course ° J m applying thebacterial methodmixture. means the living bacteria may then be got rid of by filtering mus be adopted for suitably diluting the through a filter of unglazed porcelain, whose pores are