v.] METHODS OF INOCULATION. 27 



species ; in some instances it is, however, extremely. difficult 

 to isolate after this method. 



The method of " dilution " means diluting the culture-fluid 

 containing the various species to a very large extent with some 

 sterile indifferent fluid, such as well-boiled saline solution of 

 0'6 per cent., and then inoculating new tubes with a droplet 

 of this greatly diluted material. For this purpose draw into a 

 rather large pipette a tiny droplet of the old culture-fluid, 

 then pass the pointed end of this pipette into a test-tube or 

 flask (plugged) containing well -boiled saline solution, and 

 draw up a quantity of this solution so as to greatly dilute 

 (1000- 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 inocu- 

 lation 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 or- 

 ganisms, then a droplet of this is placed into a large flask 

 containing the well-boiled saline solution, so that a dilution 

 of 1 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. 



2. Inoculations with Blood, Juices, and Tissues. To establish 

 a cultivation from blood of a dead animal, cut open the thorax 

 by removing the sternum with clean scissors, cut open the peri- 

 cardial sac, pierce with the pointed end of a fresh capillary 

 pipette the wall of the right ventricle or right auricle, and 

 allow a drop or two of blood to ascend into the pipette, or if a 

 larger quantity is required suck it up. Withdraw the pipette 

 aiid inoculate new culture-tubes as above.. Or, if blood of a 



