24 APPLIED BACTERIOLOGY 



culture and other media germ-free by exposing them to a 

 temperature of not more than 60° to 70° C. ( = 140° to 

 158° P.) for several times in succession, the operation ex- 

 tending over at least three days. 



By the first heating the adulc bacteria are killed, the 

 spores only remaining alive; the liquid is then kept at 

 about 20° to 25° C. ( = 68° to 77° F.) for about twelve to 

 twenty hours, to allow the spores to germinate, and then 

 again heated. All the spores that have developed into 

 full-grown bacteria are thus killed, and in case some of the 

 spores should not have developed, the process is repeated 

 again on one or more successive days. 



But, as has been pointed out by Miquel, absolutely 

 certain results are not to be attained by this method, as 

 some spores take days, or even weeks, to germinate. There 

 is, therefore, always the chance that some such spores may 

 be present, and may ultimately develop in a medium that 

 was believed to be sterile. 



The difference which exists between the resistance offered 

 by organisms and their spores to heat can be made use of 

 to obtain pure cultures of some spore-bearing organisms. 

 To obtain, for instance, a pure culture of the hay bacillus 

 {Bacillus suhtilis), the spores of which resist boiling water, 

 the following method can be employed : Hay is left in water 

 for twenty-four hours ; the resulting infusion is strained, 

 and one part of the liquid is diluted with ten parts of water. 

 A flask is three-quarters filled with this liquid, and the neck 

 of the flask is plugged with cotton-wool. The contents of 

 the flask are now heated to boiling, the liquid then being 

 allowed to gently simmer for an hour. In this manner all 

 other ordinary organisms and their spores are killed, the 

 spores of the hay bacillus being alone able to withstand the 

 heat of boiling-water for this length of time. These spores, 

 when the liquid is allowed to stand by for a day or two. 



