APPENDIX A 119 



temperature of 100° C. The time required for the chamber to heat 

 up depends upon the amount of material which it contains. 



When media in test tubes are to be sterilized, the tubes should 

 always be held in wire baskets which permit the easy entrance of 

 steam. If tin cups or beakers are used, it seems to be almost impos- 

 sible to dislodge a layer of cooler air from the bottom of the cup, 

 and consequently sterilization is onlj- partial. 



When the steam first enters the chamber of the sterilizer, conden- 

 sation takes place, and enough water often drops from the roof to 

 soak the cotton plugs. To guard against this, one may wait until the 

 interior of the sterilizer has reached a temperature of 100° before 

 introducing the objects to be sterilized, or the cotton plugs may be 

 covered by tj-ing manila or parchment paper over the top of the 

 wire baskets. When flasks of media are being sterilized, small beakers 

 may be inverted over the cotton plugs. Such cotton plugs as are 

 found water-soaked when the media are rfemoved from the sterilizer 

 should be replaced by new plugs and the vessels resterilized. 



Circulating steam at 100° C. will kill the vegetative forms of 

 microorganisms in fifteen to twenty minutes, but spores will survive 

 much longer heating. The success of discontinuous, or fractional, 

 sterilization was demonstrated by Tyndall. In general practice, the 

 media are sterilized for twenty to thirty minutes on three successive 

 days. In the intervals between sterilizations the media should be 

 kept at temperatures favorable for bacterial growth, in order that 

 the resting spores may germinate and pass into the vegetative stage, 

 and thus be killed at the next sterilization. Even with this treat- 

 ment the spores of anaerobic bacteria in the superficial layers of the 

 media may not develop, owing to the access of oxygen, and so may 

 survive the process of sterilization. 



Steam sterilization under pressure. The autoclave is a strong-walled 

 steam chest which maj' be tightly closed. In it the steam is used 

 under pressure, and temperatures above 100° C. are obtained. By 

 this means both the vegetative forms and the bacterial spores are 

 killed, and the necessity for discontinuous sterilization disappears. 



The type of autoclave in most common use is a heavy-walled 

 copper cylinder set in a frame above a large gas burner. The upper 

 end has a close-fitting lid which clamps on with six turnscrews. 

 The cover is provided with a steam-pressure gauge, a thermometer, 

 a stopcock, and a safety valve. A rack which fits the inside of the 

 autoclave provides one or two shelves for holding the vessels of media. 



