58 METHODS OF CULTIVATION OF BACTERIA. 



the incubator in the upright position and must be handled care- 

 fully so that the condensation water, which always is present in 

 incubated agar tubes, may not run over the surface. Agar, 

 poured out in a Petri's dish and allowed to stand till firm, may be 

 used instead of successive tubes. Here a sufficient number of 

 strokes can be made in one dish. Sloped blood-serum tubes may 

 be used instead of agar. The method is rapid and easy and 

 gives good results. 



Separation of Pathogenic Bacteria by Inoculation of Ani- 

 mals. It is found difficult and often impossible to separate by 

 ordinary plate methods certain pathogenic organisms, such as 

 B. tuberculosis, B. mallei, and the pneumococcus, when such 

 occur in conjunction with other bacteria. These grow best on 

 special media, and the first two (especially the tubercle bacillus) 

 grow so slowly that the other organisms present outgrow them, 

 cover the whole plates, and make separation impossible. The 

 method adopted in such cases is to inoculate an animal with the 

 mixture of bacilli, wait until the particular disease develops, kill 

 the animal, and with all aseptic precautions (vide p. 120) inocu- 

 late tubes of suitable media from characteristic lesions situated 

 away from the seat of inoculation, e.g. from spleen in the case 

 of B. tuberculosis, spleen or liver in the case of B. mallei, and 

 heart blood in the case of pneumococcus. 



Separation by killing Non-spored Forms by Heat. This is 

 a method which has a limited application. As has been said, 

 the spores of a bacterium resist heat more than the vegetative 

 forms. When a mixture contains spores of one bacterium and 

 vegetative forms of this and other bacteria, then if the mixture 

 be boiled for a few minutes all the vegetative forms will be 

 killed, while the spores will remain alive and will develop sub- 

 sequently. This method can be easily tested in the case of 

 cultivating B. subtilis from hay infusion. A little chopped-up 

 hay is placed in a flask of water, which is boiled for about ten 

 minutes. On this being allowed to cool and stand, in a day or 

 two a scum forms on the surface, which is found to be a pure 

 culture of the bacillus subtilis. The method is also often used 

 to aid in the separation of B. tetani, vide infra. 



