56 METHODS OF CULTIVATION OF BACTERIA 



of diphtheritic membrane. In the case of a tissue, for example, 

 a small portion entangled in the loop of a platinum needle is 

 stroked in successive parallel longitudinal strokes on sloped 

 agar, the same aspect being brought in contact with the agar in 

 all the strokes. Three strokes may be made on each tube, and 

 three tubes are usually sufficient. In this process the organisms 

 on the surface of the tissue are gradually rubbed off, and when 

 growth has taken place it will be found that 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 diphtheritic membrane, putrefactive organisms may 

 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 incubator in the upright position and must be 

 handled carefully 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 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 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. 123) 

 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. When a mixture contains spores of one bacterium and 

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



