DIFGO MANUAL 



19 



If all the air is not removed from the sterilizing chamber, which condition is 

 best shown by use of a thermometer in the exhaust line of the autoclave, an en- 

 tirely different pressure-temperature relationship exists. Through the courtesy of 

 Dr. F. W. Tanner 2 we are able to reproduce the following chart which plainly 

 shows the actual temperature in the autoclave when the air is not completely 

 exhausted. 



Effect of Entrapped Air on Temperature of Autoclave 



•250i 



PER CENT AIR IN SItAM, 



When the operator is assured that all the air is replaced by steam, which is best 

 indicated by a thermometer placed in the exhaust line, the outlet valve of the 

 autoclave is closed and the steam pressure is raised to 15 pounds. When the ther- 

 mometer indicates a temperature of 121°C. heating is continued for 15 minutes. 

 A maximum of 15 minutes is recommended for the sterilization of carbohydrate 

 media in tubes to be used for fermentation studies. After the sterilization period 

 has been completed, the source of steam is cut off and the autoclave is allowed 

 to return to atmospheric pressure. Pressure should not drop too rapidly or the 

 media will boil over, blowing the plugs from the tubes or flasks. Pressure should, 

 however, drop rapidly enough to prevent excessive exposure of the media to heat 

 after the sterilization period. Ordinarily about 8, and not more than 1 2, minutes 

 should be required for the usual bacteriological laboratory autoclave to reach 

 atmospheric pressure without danger of prolonged heating. The media should be 

 removed from the autoclave shortly after sterilization and should not be per- 

 mitted to remain in the autoclave for any appreciable length of time after the 

 sterilization period. 



For the sterilization of coagulable material such as serum, see the method given 

 for Bacto-LoeflPier Blood Serum, page 128. 



Oversterilization or prolonged heating will change the composition of the 

 medium. For example, in our laboratories we have shown that Phenol Red Lac- 

 tose Broth which has been sterilized for 15 minutes at 15 pounds pressure 

 (121°C.), or sterilized by filtration, produces no demonstrable amount of acid 

 when inoculated with Salmonella typhosa. The same lot of medium sterilized for 

 30 or 45 minutes at 121°C. showed appreciable acid production under the same 

 conditions of testing. This demonstrates that oversterilization resulted in a break- 

 down of the lactose. 



Agar media on prolonged sterilization or heating are apt to show a precipitate. 



