TEMPERATURE 301 



12. Pipette the remaining 4 c.c. of the suspension 

 into a culture flask containing 250 c.c. of nutrient 

 bouillon, and incubate. 



13. Observe these cultivations from day to day. 

 "No growth" must not be recorded as final until after 

 the completion of seven days' incubation. 



14. Extend these observations to the remaining 

 tubes of the series, but varying the conditions so that 

 each tube is exposed to a temperature 2 C. higher 

 than the immediately preceding one i. e., 42 C., 44 

 C., 46 C., and so on. 



15. Note that temperature, after exposure to which 

 no growth takes place up to the end of seven days' 

 incubation, = the thermal death-point. 



1 6. If greater accuracy is desired, a second series 

 of tubes may be prepared and exposed for ten minutes 

 to fixed temperatures varying only 0.5 C., through 

 a range of 5 C. on either side of the previously observed 

 death-point. 



Moist Spores : The thermal death-point in the case 

 of spores is that time exposure to a fixed temperature of 

 100 C. necessary to effect the death of all the spores 

 present in a suspension. 



NOTE. If it is desired to retain the time constant 10 minutes 



and investigate the temperature necessary to destroy the spores, 

 varying amounts of calcium chloride must be added to the. water 

 in the bath, when the boiling-point will be raised above 100 C. 

 according to the percentage of calcium in solution. In such case 

 use the bath figured on page 227 the bath figured on page 299 can 

 only be used if the capsule; is first removed. 



It is determined in the following manner 



Apparatus Required: 



Steam-can fitted with a delivery tube and a large bore safety- 

 valve tube. 



Water-bath at 100 C. 



Erlenmeyer flask, 500 c.c. capacity, containing 140 c.c. sterile 

 normal saline solution and fitted with rubber stopper perforated 

 with four holes. 



The rubber stopper is fitted as follows: 



