62 METHODS OF CULTIVATION OF BACTERIA 



contained in a fairly solid tissue or substance, such as a piece 

 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 in 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 is always 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 grow so slowly that the other 

 organisms present may outgrow them, cover the whole plates, and 

 make separation difficult. 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. 147) 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 



