SEPARATION OF BACTERIA BY SPECIAL METHODS, 63 



in the later strokes the colonies are less numerous than in 

 the earlier, and sufficiently far apart to enable parts of 

 them to be picked off without the needle touching any but 

 one colony. When, as in the case of diphtheria membrane, 

 putrefactive organisms are likely to be present on the surface 

 of the tissue, these can be in great part removed by washing 

 it well in cold water previously sterilised (vide Diphtheria). 

 In the case of liquids, the loop is charged and similarly 

 stroked. Tubes thus inoculated must be put in the in- 

 cubator 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 capsule and allowed to stand 

 till firm, may be used instead of successive tubes. Here a 

 sufficient number of strokes can be made in one capsule. 

 Sloped inspissated 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 

 Animals. 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 these 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 it, and with all aseptic precautions (vide p. 115) inoculate 

 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. If there is a mixture containing spores of 



