EXPERIMENTS IN STERILIZATION 329 



heated for ten minutes, though the colonies will be fewer 

 in number. 



The thirty-minute tube may or may not give one or two 

 colonies of the same organism. 



The tube which has been heated for one hour is usually 

 sterile. 



The bouillon tubes from the first and second tubes which 

 were heated will usually show the presence of only one 

 organism the bacillus which gave rise to the pellicle- 

 formation in the original mixture. This organism is bacillus 

 subtilis. It is especially adapted to the study of those various 

 degrees of resistance to heat that spore-forming bacteria 

 exhibit at different stages of their development. 



Inoculate about 100 c.c. of sterilized bouillon with a very 

 small quantity of a pure culture of this organism, and allow 

 it to stand in a warm place for about six hours. Now subject 

 this culture to the action of steam for five minutes; it will 

 be seen that sterilization, as a rule, is complete. 



Treat in the same way a second flask of bouillon, inocu- 

 lated in the same way with the same organism, but after 

 having stood in a warm place for from forty-eight to seventy- 

 two hours that is, until spores have formed and it will 

 be found that sterilization is not complete : the spores of 

 this organism have resisted the action of steam for five 

 minutes. 



To determine if sterilization is complete always resort 

 to the culture methods, as the macroscopic and microscopic 

 methods are deceptive; cloudiness of the media or the 

 presence of bacteria microscopically does not always signify 

 that organisms possess the property of life. 



Inoculate in the same way a third flask of bouillon with 

 a very small drop from one of the old cultures upon which 

 the pellicle has formed; mix it well and subject it to the 



