FRACTIONAL STERILISATION 25 



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 subtilis), 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, 

 begin to develop vigorously, the hay infusion which contains 

 the spores being a most favourable medium for the growth 

 of the hay bacillus. As the cotton- wool plug prevents the 

 entrance of other organisms, a pure culture of the hay 

 bacillus is thus obtained. 



A similar method, using regulated temperatures, is em- 

 ployed to obtain pure cultures of the tetanus bacillus. 



(3) Action of Cold. — Micro-organisms are extremely resist- 

 ant with regard to cold. Frosch, in 1887, exposed various 

 cultures to a temperature of —87° C, which he obtained by 

 means of liquid carbon dioxide, and found that most of the 

 organisms experimented upon multiplied on being placed 

 again under favourable conditions. Prudden has recently 

 , made some extended experiments on the influence of 

 freezing. He found that while some organisms withstood 

 the action of cold for a long time, others failed to grow. 



