II.] PREPARATION OF CULTURE MATERIAL. 19 



(Fig. 3), and this is allowed to stand for one night. Next 

 day the boiling is repeated for half an hour or more in the 

 same manner as before. If the meat has been fresh and the 

 vessels and cotton-wool have been sterile, twice boiling is 

 found sufficient to destroy every impurity. But to make 

 sure, the broth is placed in the incubator and kept there for 

 twenty-four hours at a temperature of 32 to 38 C., and then 

 boiled on the next day for half an hour in the usual way. 

 The supposition is made, that if by any chance after twice 

 boiling the broth it should contain unchanged spores of 

 bacilli the only organisms that will resist boiling, although 

 they do not resist boiling for more than half an hour the 

 spores would germinate into bacilli when kept for twenty- 

 four hours in the incubator at 32 to 38, and these would 

 then be killed by the third boiling. As a matter of fact I 

 have not as a rule found any contaminating germs survive 

 the second boiling. It is of course to be borne in mind 

 that during the first as well as second and subsequent boiling 

 the cotton-wool plug is not removed from the mouth of the 

 flask, but is only raised out half its length from the neck. 

 The cotton-wool and the cotton-wool cap and beaker are 

 replaced immediately or simultaneously with the turning off 

 of the burner. This broth so prepared is placed in the 

 incubator at 32 to 38 C. and kept there from one to three 

 weeks. If, as is generally the case, it remains limpid, it is 

 considered completely sterile. 



2. Peptone and Sugar Solution. Beef peptone (Savory 

 and Moore's) is dissolved in distilled water, over a burner, 

 to the amount of about 2 per cent. ; to the solution is added 

 cane-sugar to the amount of about i per cent. ; so that every 

 100 ccm. of the fluid contains two grammes of peptone and 

 one gramme of sugar. When dissolved it is well neutralised 

 and then filtered (the vessels being of course also in this, as 



C 2 



