STERILIZATION BY STEAM AND BY HOT AIE. 231 



minutes will present colonies in moderate numbers, but, 

 as a rule, they will represent but a single organism. 

 Study this organism in pure cultures. 



The same may be predicted for the tube which has 

 been 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 our original mixture. This organ- 

 ism is the bacillus subtilis, and will serve as an object 

 upon which to study the difference in resistance toward 

 steam between the vegetative and spore stages of the 

 same organism. 



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, in- 

 oculated 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 the 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 micro- 



