v.] METHODS OF INOCULATION. 41 



greatly dilute (looo-fold or more) the droplet of culture- 

 fluid, and with this inoculate then a series of new culture- 

 tubes containing different nourishing material, using always 

 only a trace for inoculation. In this way it is probable that, 

 owing to the great dilution, the trace of a droplet of this 

 mixture used for the new inoculation contains only one 

 species. Using a series of new culture-tubes and inoculating 

 them thus, after twenty-four hours of incubation it will be 

 found that some tubes have not received any seed, others 

 only one species. If it be required to dilute the original fluid 

 greatly, say if it teems with different organisms, then a droplet 

 of this is placed into a large flask containing the well-boiled 

 saline solution, so that a dilution of i in 1,000,000 or more 

 can be effected. 



The two methods, i.e. that of fractional culture and of 

 dilution, may be successfully combined in this way : from 

 the first or second new culture, established after the method 

 of fractional cultivation, in which after twenty-four or thirty- 

 six hours one species greatly predominates, draw out with a 

 large capillary pipette a droplet, and dilute this to a great 

 extent with the saline solution, as described above, and now 

 inoculate with a trace of this mixture a new culture-tube. 

 Or, if after twenty-four hours' incubation the microscope 

 reveals in this further culture more than one species, continue 

 the process of dilution and inoculation for a further generation. 

 Thus it is possible to obtain cultures of only one species, 

 although the original fluid contained several species of 

 organisms. 



One of the best methods for isolation is that of the plate- 

 cultivation introduced by Koch in connection with the 

 isolation of the choleraic comma bacilli. A test-tube 

 containing sterile nutritive gelatine as above prepared is 

 liquefied by gentle heat, best by being kept in water of about 



