2l8 PATHOGENIC BACTERIA. The best method of obtaining a culture is to inoculate a guinea-pig with tuberculous material, allow an artificial tuberculosis to develop, kill the animal after a couple of months, and make the cultures from the centre of one of the tuberculous glands. Of course many technical difficulties must be over- come. The tuberculous material used for inoculaticm may be sputum, injected beneath the skin by a hypo- dermic syringe. The animal is allowed to live for a month or six weeks, then killed. The autopsy is per- formed according to directions already given. A large lymphatic gland with softened contents or a nodule in the spleen being selected for the culture, an incision is made into it with a sterile knife, or a rigid sterile platinum wire is introduced; some of the contents are removed and planted upon blood-serum. After receiving the in- oculated material the tubes are closed, either by a rub- ber cap placed over the cotton stopper, which is cut off and pushed in, or by a rubber cork above the cotton, the idea of this rubber corking being simply to prevent evaporation. The tubes must be kept in an incubator at the temperature of 37-38° C. Kitasato has published a method by which Koch has been able to secure the tubercle bacillus in pure culture from sputum. After carefully cleansing the mouth the patient is allowed to expectorate into a sterile Petri dish. By this method the contaminating bacteria from the mouth and the receptacle are excluded, and the expecto- rated material is made to contain only such bacteria as were present in the lungs. The material is carefully washed a great many times in renewed distilled sterile water until all bacteria not enclosed in the muco-purulent material are removed; it is then carefully opened with sterile instruments, and the culture-medium—glycerin agar-agar or blood-serum—is inoculated from the centre. Kitasato has been able by this method to demonstrate that many of the bacilli ordinarily present in tubercular sputum are dead, although they continue to stain well.